Search results for the GEO ID: GSE7292  |
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GSM ID | GPL ID |
Select for analysis |
Title |
Source name |
Description |
Characteristics |
GSM174821 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab control 1
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 3
|
untreated immunomagnetically selected primary CD19+ B-cells
|
| Sample_geo_accession | GSM174821
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | untreated CD19+ B-cells
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174821/suppl/GSM174821.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174822 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab control 2
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 1
|
untreated immunomagnetically selected primary CD19+ B-cells
|
| Sample_geo_accession | GSM174822
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | untreated CD19+ B-cells
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174822/suppl/GSM174822.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174823 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab control 3
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of a healthy donor
|
untreated immunomagnetically selected primary CD19+ B-cells
|
| Sample_geo_accession | GSM174823
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | untreated CD19+ B-cells
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174823/suppl/GSM174823.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174824 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab control 4
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 3
|
untreated immunomagnetically selected primary CD19+ B-cells
|
| Sample_geo_accession | GSM174824
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | untreated CD19+ B-cells
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174824/suppl/GSM174824.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174825 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab control 5
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of a healthy donor
|
untreated immunomagnetically selected primary CD19+ B-cells
|
| Sample_geo_accession | GSM174825
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | untreated CD19+ B-cells
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174825/suppl/GSM174825.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174826 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab control 6
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 3
|
untreated immunomagnetically selected primary CD19+ B-cells
|
| Sample_geo_accession | GSM174826
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | untreated CD19+ B-cells
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174826/suppl/GSM174826.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174827 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab control 7
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 4
|
untreated immunomagnetically selected primary CD19+ B-cells
|
| Sample_geo_accession | GSM174827
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | untreated CD19+ B-cells
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174827/suppl/GSM174827.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174828 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab control 8
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 2
|
untreated immunomagnetically selected primary CD19+ B-cells
|
| Sample_geo_accession | GSM174828
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | untreated CD19+ B-cells
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174828/suppl/GSM174828.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174867 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab 100 µCi, 46 min, sample 1
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 3
|
Bi-213-CD20-Rituximab irradiated primary CD19+ B-cells
|
| Sample_geo_accession | GSM174867
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The alpha-emitter Bi-213 was conjugated with CHX-A’’-DTPA-chelated CD20 antibody Rituximab.
| | Sample_treatment_protocol_ch1 | Subsequently the purified Bi-213-labelled antibody was added to the immunomagnetically selected primary CD19+ B-cells.
| | Sample_treatment_protocol_ch1 | A constant number of 1x106 CD19+ B-cells were incubated in a final volume of 2 ml with an activity of Bi-213-CD20 of 100 µCi for 46 minutes (one half life time) at 37°C, 5% CO2, 95% humidity.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174867/suppl/GSM174867.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174875 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab 100 µCi, 46 min, sample 2
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 3
|
Bi-213-CD20-Rituximab irradiated primary CD19+ B-cells
|
| Sample_geo_accession | GSM174875
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The alpha-emitter Bi-213 was conjugated with CHX-A’’-DTPA-chelated CD20 antibody Rituximab.
| | Sample_treatment_protocol_ch1 | Subsequently the purified Bi-213-labelled antibody was added to the immunomagnetically selected primary CD19+ B-cells.
| | Sample_treatment_protocol_ch1 | A constant number of 1x106 CD19+ B-cells were incubated in a final volume of 2 ml with an activity of Bi-213-CD20 of 100 µCi for 46 minutes (one half life time) at 37°C, 5% CO2, 95% humidity.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174875/suppl/GSM174875.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174876 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab 100 µCi, 46 min, sample 3
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 2
|
Bi-213-CD20-Rituximab irradiated primary CD19+ B-cells
|
| Sample_geo_accession | GSM174876
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The alpha-emitter Bi-213 was conjugated with CHX-A’’-DTPA-chelated CD20 antibody Rituximab.
| | Sample_treatment_protocol_ch1 | Subsequently the purified Bi-213-labelled antibody was added to the immunomagnetically selected primary CD19+ B-cells.
| | Sample_treatment_protocol_ch1 | A constant number of 1x106 CD19+ B-cells were incubated in a final volume of 2 ml with an activity of Bi-213-CD20 of 100 µCi for 46 minutes (one half life time) at 37°C, 5% CO2, 95% humidity.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174876/suppl/GSM174876.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174877 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab 100 µCi, 46 min, sample 4
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 2
|
Bi-213-CD20-Rituximab irradiated primary CD19+ B-cells
|
| Sample_geo_accession | GSM174877
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The alpha-emitter Bi-213 was conjugated with CHX-A’’-DTPA-chelated CD20 antibody Rituximab.
| | Sample_treatment_protocol_ch1 | Subsequently the purified Bi-213-labelled antibody was added to the immunomagnetically selected primary CD19+ B-cells.
| | Sample_treatment_protocol_ch1 | A constant number of 1x106 CD19+ B-cells were incubated in a final volume of 2 ml with an activity of Bi-213-CD20 of 100 µCi for 46 minutes (one half life time) at 37°C, 5% CO2, 95% humidity.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174877/suppl/GSM174877.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174878 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab 100 µCi, 46 min, sample 5
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 2
|
Bi-213-CD20-Rituximab irradiated primary CD19+ B-cells
|
| Sample_geo_accession | GSM174878
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The alpha-emitter Bi-213 was conjugated with CHX-A’’-DTPA-chelated CD20 antibody Rituximab.
| | Sample_treatment_protocol_ch1 | Subsequently the purified Bi-213-labelled antibody was added to the immunomagnetically selected primary CD19+ B-cells.
| | Sample_treatment_protocol_ch1 | A constant number of 1x106 CD19+ B-cells were incubated in a final volume of 2 ml with an activity of Bi-213-CD20 of 100 µCi for 46 minutes (one half life time) at 37°C, 5% CO2, 95% humidity.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174878/suppl/GSM174878.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174879 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab 100 µCi, 46 min, sample 6
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 1
|
Bi-213-CD20-Rituximab irradiated primary CD19+ B-cells
|
| Sample_geo_accession | GSM174879
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The alpha-emitter Bi-213 was conjugated with CHX-A’’-DTPA-chelated CD20 antibody Rituximab.
| | Sample_treatment_protocol_ch1 | Subsequently the purified Bi-213-labelled antibody was added to the immunomagnetically selected primary CD19+ B-cells.
| | Sample_treatment_protocol_ch1 | A constant number of 1x106 CD19+ B-cells were incubated in a final volume of 2 ml with an activity of Bi-213-CD20 of 100 µCi for 46 minutes (one half life time) at 37°C, 5% CO2, 95% humidity.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174879/suppl/GSM174879.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174880 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab 100 µCi, 46 min, sample 7
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 4
|
Bi-213-CD20-Rituximab irradiated primary CD19+ B-cells
|
| Sample_geo_accession | GSM174880
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The alpha-emitter Bi-213 was conjugated with CHX-A’’-DTPA-chelated CD20 antibody Rituximab.
| | Sample_treatment_protocol_ch1 | Subsequently the purified Bi-213-labelled antibody was added to the immunomagnetically selected primary CD19+ B-cells.
| | Sample_treatment_protocol_ch1 | A constant number of 1x106 CD19+ B-cells were incubated in a final volume of 2 ml with an activity of Bi-213-CD20 of 100 µCi for 46 minutes (one half life time) at 37°C, 5% CO2, 95% humidity.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174880/suppl/GSM174880.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174881 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab 200 µCi, 46 min, sample 1
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 3
|
Bi-213-CD20-Rituximab irradiated primary CD19+ B-cells
|
| Sample_geo_accession | GSM174881
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The alpha-emitter Bi-213 was conjugated with CHX-A’’-DTPA-chelated CD20 antibody Rituximab.
| | Sample_treatment_protocol_ch1 | Subsequently the purified Bi-213-labelled antibody was added to the immunomagnetically selected primary CD19+ B-cells.
| | Sample_treatment_protocol_ch1 | A constant number of 1x106 CD19+ B-cells were incubated in a final volume of 2 ml with an activity of Bi-213-CD20 of 200 µCi for 46 minutes (one half life time) at 37°C, 5% CO2, 95% humidity.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174881/suppl/GSM174881.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174882 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab 200 µCi, 46 min, sample 2
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 3
|
Bi-213-CD20-Rituximab irradiated primary CD19+ B-cells
|
| Sample_geo_accession | GSM174882
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The alpha-emitter Bi-213 was conjugated with CHX-A’’-DTPA-chelated CD20 antibody Rituximab.
| | Sample_treatment_protocol_ch1 | Subsequently the purified Bi-213-labelled antibody was added to the immunomagnetically selected primary CD19+ B-cells.
| | Sample_treatment_protocol_ch1 | A constant number of 1x106 CD19+ B-cells were incubated in a final volume of 2 ml with an activity of Bi-213-CD20 of 200 µCi for 46 minutes (one half life time) at 37°C, 5% CO2, 95% humidity.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174882/suppl/GSM174882.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174892 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab 200 µCi, 46 min, sample 3
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 4
|
Bi-213-CD20-Rituximab irradiated primary CD19+ B-cells
|
| Sample_geo_accession | GSM174892
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The alpha-emitter Bi-213 was conjugated with CHX-A’’-DTPA-chelated CD20 antibody Rituximab.
| | Sample_treatment_protocol_ch1 | Subsequently the purified Bi-213-labelled antibody was added to the immunomagnetically selected primary CD19+ B-cells.
| | Sample_treatment_protocol_ch1 | A constant number of 1x106 CD19+ B-cells were incubated in a final volume of 2 ml with an activity of Bi-213-CD20 of 200 µCi for 46 minutes (one half life time) at 37°C, 5% CO2, 95% humidity.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174892/suppl/GSM174892.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174893 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab 200 µCi, 46 min, sample 4
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 4
|
Bi-213-CD20-Rituximab irradiated primary CD19+ B-cells
|
| Sample_geo_accession | GSM174893
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The alpha-emitter Bi-213 was conjugated with CHX-A’’-DTPA-chelated CD20 antibody Rituximab.
| | Sample_treatment_protocol_ch1 | Subsequently the purified Bi-213-labelled antibody was added to the immunomagnetically selected primary CD19+ B-cells.
| | Sample_treatment_protocol_ch1 | A constant number of 1x106 CD19+ B-cells were incubated in a final volume of 2 ml with an activity of Bi-213-CD20 of 200 µCi for 46 minutes (one half life time) at 37°C, 5% CO2, 95% humidity.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174893/suppl/GSM174893.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
GSM174894 | GPL201 |
|
Alpha-irradiated primary CD19+ B-cells with the radioimmuno-conjugate Bi-213-CD20-Rituximab 200 µCi, 46 min, sample 5
|
Primary CD19+ B-Cells
|
Immunomagnetically selected from peripheral blood of healthy donor 1
|
Bi-213-CD20-Rituximab irradiated primary CD19+ B-cells
|
| Sample_geo_accession | GSM174894
| | Sample_status | Public on Jul 01 2007
| | Sample_submission_date | Mar 13 2007
| | Sample_last_update_date | Mar 20 2007
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Homo sapiens
| | Sample_taxid_ch1 | 9606
| | Sample_treatment_protocol_ch1 | The alpha-emitter Bi-213 was conjugated with CHX-A’’-DTPA-chelated CD20 antibody Rituximab.
| | Sample_treatment_protocol_ch1 | Subsequently the purified Bi-213-labelled antibody was added to the immunomagnetically selected primary CD19+ B-cells.
| | Sample_treatment_protocol_ch1 | A constant number of 1x106 CD19+ B-cells were incubated in a final volume of 2 ml with an activity of Bi-213-CD20 of 200 µCi for 46 minutes (one half life time) at 37°C, 5% CO2, 95% humidity.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | RNA of alpha irradiated K422 cells was extracted using the RNeasy
| | Sample_extract_protocol_ch1 | Micro- Kit (Qiagen, Santa Clarita, CA, USA Cat. No. 74004). All buffers and
| | Sample_extract_protocol_ch1 | solutions are part of the kit and were applied according to the manufacturer’s instructions.
| | Sample_extract_protocol_ch1 | Briefly:
| | Sample_extract_protocol_ch1 | Alpha irradiated cells suspended in 350 µl lysis buffer were incubated with 350 µl 70%
| | Sample_extract_protocol_ch1 | ethanol.
| | Sample_extract_protocol_ch1 | 700 µl suspension was put on RNeasy MinElute- column and spun down for one
| | Sample_extract_protocol_ch1 | minute with 10,000 rpm.
| | Sample_extract_protocol_ch1 | One washing step with 350 µl RW1 buffer
| | Sample_extract_protocol_ch1 | DNase- digestion (10 µl DNase+70 µl RDD buffer for each sample) for 15 minutes at room temperature.
| | Sample_extract_protocol_ch1 | 2 washing steps with 350 µl RW1- buffer and one step with 500 µl RPE- buffer.
| | Sample_extract_protocol_ch1 | 500 µl of 80% ethanol were put on RNeasy MinElute- column and spun down for 2
| | Sample_extract_protocol_ch1 | minutes with 10,000 rpm.
| | Sample_extract_protocol_ch1 | Removal of ethanol: column was spun down for 5 minutes with 13,000 rpm.
| | Sample_extract_protocol_ch1 | Elution of RNA with 14 µl of RNAse free water.
| | Sample_extract_protocol_ch1 | Quality and quantity control of the RNA using a Agilent 2100 Bioanalyzer
| | Sample_extract_protocol_ch1 | (Agilent Technologies, Palo Alto CA) and the NanoDrop ND-1000 (PeqLab
| | Sample_extract_protocol_ch1 | Biotechnologie
| | Sample_extract_protocol_ch1 | GmbH, Erlangen). Purified RNA was stored at -80°C.
| | Sample_label_ch1 | Biotin/Streptavidin-PE
| | Sample_label_protocol_ch1 | Amplification, reverse transcription and labelling of the
| | Sample_label_protocol_ch1 | sample.
| | Sample_label_protocol_ch1 | For amplification of the RNA of the irradiated cells, the MessageAmp™ aRNA Kit (Ambion,
| | Sample_label_protocol_ch1 | Austin, TX, USA) was used.
| | Sample_label_protocol_ch1 | 500 ng of isolated RNA were transcribed into cDNA.
| | Sample_label_protocol_ch1 | First strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 11 µl (RNA or RNA and dH2O) Sample
| | Sample_label_protocol_ch1 | 1 µl Oligo(dT)Primer
| | Sample_label_protocol_ch1 | 70°C incubation for 10 minutes, put on ice and incubated with the reaction kit
| | Sample_label_protocol_ch1 | (stated below).
| | Sample_label_protocol_ch1 | Reaction kit:
| | Sample_label_protocol_ch1 | 2 µl 10x First Strand Buffer
| | Sample_label_protocol_ch1 | 1 µl Ribonuclease Inhibitor
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | Adding 1µl Reverse transcriptase and 2 h incubation at 42°C
| | Sample_label_protocol_ch1 | Second strand cDNA- Synthesis:
| | Sample_label_protocol_ch1 | 63 µl Nuclease-free Water
| | Sample_label_protocol_ch1 | 10 µl 10x Second Strand Buffer
| | Sample_label_protocol_ch1 | 4 µl dNTP Mix
| | Sample_label_protocol_ch1 | 1 µl RNase H
| | Sample_label_protocol_ch1 | For second strand sythesis 2 µl of DNA- Polymerase are added followed by a 2 h
| | Sample_label_protocol_ch1 | Incubation step at 42°C.
| | Sample_label_protocol_ch1 | Purification of cDNA:
| | Sample_label_protocol_ch1 = Final elution volume | 16 µl and storage for 12 h at -20°C
| | Sample_label_protocol_ch1 | Labelling with in vitro transcription using biotin labelled nucleotides:
| | Sample_label_protocol_ch1 | Labeling mix:
| | Sample_label_protocol_ch1 | 4µl biotinylated dNTPs (Bio Array High Yield RNA Transcript Labelling Kit,
| | Sample_label_protocol_ch1 | Enzo Life Sciences, Farmingdale)
| | Sample_label_protocol_ch1 | 4 µl T7 10x reaction buffer
| | Sample_label_protocol_ch1 | 12 µl RNase free water
| | Sample_label_protocol_ch1 | The labelling mix is incubated with the sample and 4 µl of the enzyme mix is
| | Sample_label_protocol_ch1 | added followed by an incubation period of 5 hours at 37°C. The amplified RNA
| | Sample_label_protocol_ch1 | is purified and is eluted in a final volume of 96 µl.
| | Sample_label_protocol_ch1 | Quality control of aRNA:
| | Sample_label_protocol_ch1 | The concentration and purity of aRNA is measured using the NanoDrop ND-1000
| | Sample_label_protocol_ch1 | (PeqLab Biotechnologie GmbH, Erlangen) measuring absorptions at 260 and 280nm
| | Sample_label_protocol_ch1 | The OD 260/OD 280 aRNA was between 1.8 and 2,1. To exclude
| | Sample_label_protocol_ch1 | degradation and/or contamination RNA was analyzed again using an Agilent 2100
| | Sample_label_protocol_ch1 | Bioanalyzer (Agilent Technologies, Palo Alto CA).
| | Sample_label_protocol_ch1 | Fragmentation:
| | Sample_label_protocol_ch1 | For optimal hybridization with the complementary oligonucleodid sequences, a
| | Sample_label_protocol_ch1 | fragmentation of the amplified RNA was performed.
| | Sample_label_protocol_ch1 | 24 µl of the sample were added to 6 µl of fragmentation buffer (200 mM
| | Sample_label_protocol_ch1 | TRIS-Acetat, ph 8.2, 500 mM KOAc, 150 mM MgOAc, Affymetrix) and incubated at
| | Sample_label_protocol_ch1 | 94°C for 35 minutes.
| | Sample_hyb_protocol | Arrays were prehybridizised for 10 minutes using 140 µl
| | Sample_hyb_protocol | MES- hybridization buffer (70,4 g MES free acid monohydrate, 193.3 g MES Sodium
| | Sample_hyb_protocol | Salt, 800 ml accugen water, pH 6.5 – 6.7) and 140 µl aqua dest. at 45°C and 60 rpm in the Gene Chip® Hybridizing oven 640 (Affymetrix).
| | Sample_hyb_protocol | After removal of the pre-hybridizing solution, the array was loaded with 250 µl
| | Sample_hyb_protocol | of the hybridizing solution (30 µl fragmented aRNA, 5 µl control-oligo B2, 15 µl
| | Sample_hyb_protocol | 20x Eukaryotic hybridizing control, 3 µl salmon sperm-DNA, 3 µl acetylic BSA,
| | Sample_hyb_protocol | 150 µl 2x MES- Hybridizing buffer and 94 µl aqua dest.).
| | Sample_hyb_protocol | Following 16-hour incubation at 45°C with permanent rotation, the hybridizing
| | Sample_hyb_protocol | solution is removed.
| | Sample_hyb_protocol | The array was loaded with 280 µl of washing buffer A (300 ml 20x SSPE, 1ml 10%
| | Sample_hyb_protocol | Tween 20, 698 ml accugen water) and a maximum of 4 hybridized arrays are put
| | Sample_hyb_protocol | into the wash station ((Gene Chip® Fluidics, Affymetrix) with buffer A and buffer B
| | Sample_hyb_protocol | (83.3 ml 12x MES, 5.2 ml 5M NaCl, 1ml 10% Tween 20, 910.5 ml accugen water)
| | Sample_hyb_protocol | using the washing protocol which is prescribed by Affymetrix (for Human Genome Focus
| | Sample_hyb_protocol = Oligonukleotid- Arrays | Midi-euk2v3).
| | Sample_hyb_protocol | During washing procedure the staining solution is prepared:
| | Sample_hyb_protocol | Staining solution:
| | Sample_hyb_protocol | 600 µl 2x Staining Buffer
| | Sample_hyb_protocol | 540 µl dH2O
| | Sample_hyb_protocol | 48 µl BSA
| | Sample_hyb_protocol | 12 µl Streptavidine-Phycoerythrin-Conjugate
| | Sample_hyb_protocol | Array was incubated with 600 µl of the staining solution after washing for 40
| | Sample_hyb_protocol | minutes followed by incubation with the antibody solution for 10 minutes:
| | Sample_hyb_protocol | Antibody solution:
| | Sample_hyb_protocol | 300 µl 2x Staining Buffer 266
| | Sample_hyb_protocol | 4 µl dH2O
| | Sample_hyb_protocol | 24 µl BSA
| | Sample_hyb_protocol | 6 µl Goat IgG
| | Sample_hyb_protocol | 3.6 µl biotinylated Anti- Streptavidin- antibody
| | Sample_hyb_protocol | After removal of the antibody solution 600 µl of the staining solution is applied again for 10 minutes followed by removal of the staining solution and washing.
| | Sample_scan_protocol | The scanning of the array was performed using a GeneArray® 2500 Scanner (Affymetrix, Software GCOS[GeneChip Operating System]). Excitation of the fluorochrome
| | Sample_scan_protocol | phycoerythrin at a wave length of 488 nm with an emission at 570 nm.
| | Sample_data_processing | Using the affy package of functions of statistical scripting
| | Sample_data_processing | language 'R' integrated into the Bioconductor project data from cel files of
| | Sample_data_processing | each sample were normalized by variance stabilizing transformations (VSN)
| | Sample_data_processing | Ref.: Huber W, von Heydebreck A, Sultmann H, et al. Bioinformatics
| | Sample_data_processing | 2002;18:96-104.
| | Sample_platform_id | GPL201
| | Sample_contact_name | Sascha,,Raschke
| | Sample_contact_laboratory | Hämatologisches Forschungslabor Geb. 23.12.00.62
| | Sample_contact_department | Klinik für Hämatologie, Onkologie und klinische Immunologie
| | Sample_contact_institute | Heinrich-Heine Universität Düsseldorf
| | Sample_contact_address | Universitätsstraße 1
| | Sample_contact_city | Düsseldorf
| | Sample_contact_zip/postal_code | 40225
| | Sample_contact_country | Germany
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM174nnn/GSM174894/suppl/GSM174894.CEL.gz
| | Sample_series_id | GSE7292
| | Sample_series_id | GSE7322
| | Sample_data_row_count | 8746
| | |
|
| |
| |
Make groups for comparisons |
(2 groups will be compared at a time) |
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| Select GSMs and click on "Add groups" |
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