Search results for the GEO ID: GSE14481  |
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|
GSM ID | GPL ID |
Select for analysis |
Title |
Source name |
Description |
Characteristics |
GSM357103 | GPL1261 |
|
E13.5 Mouse testis_129/Sv_1
|
E13.5 male genital ridges 129/Sv
|
Background Srain: 129/SV
Gender: male
Tissue: testis
Age: E13.5
|
E13.5 Mouse testis_129/Sv_1
|
| Sample_geo_accession | GSM357103
| | Sample_status | Public on Dec 31 2009
| | Sample_submission_date | Jan 02 2009
| | Sample_last_update_date | Jan 21 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Mus musculus
| | Sample_taxid_ch1 | 10090
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | 40 pairs of testes were pooled for two affymetrix arrays. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
| | Sample_label_ch1 | Biotin
| | Sample_label_protocol_ch1 | Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
| | Sample_label_protocol_ch1 | Use a starting amount of 5.0 – 40.0 mg total RNA.
| | Sample_label_protocol_ch1 | First Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
| | Sample_label_protocol_ch1 | 2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | 3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
| | Sample_label_protocol_ch1 | 4.Incubate at 42° C for 2 minutes.
| | Sample_label_protocol_ch1 | 5.Add to the tube 1 mL of Superscript II reverse transcriptase.
| | Sample_label_protocol_ch1 | 6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
| | Sample_label_protocol_ch1 | 2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
| | Sample_label_protocol_ch1 | 3.Add 2 mL [10 U] T4 DNA Polymerase.
| | Sample_label_protocol_ch1 | 4.Incubate at 16° C for 5 minutes.
| | Sample_label_protocol_ch1 | 5.Add 10 mL 0.5 M EDTA.
| | Sample_label_protocol_ch1 | Cleanup of Double-Stranded cDNA
| | Sample_label_protocol_ch1 | Phenol/Chloroform Extraction
| | Sample_label_protocol_ch1 | 1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
| | Sample_label_protocol_ch1 | 2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
| | Sample_label_protocol_ch1 | Ethanol Precipitation
| | Sample_label_protocol_ch1 | 1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
| | Sample_label_protocol_ch1 | 2.Immediately spin at maximum speed for 20 minutes at room temperature.
| | Sample_label_protocol_ch1 | 3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 5.Air dry pellet.
| | Sample_label_protocol_ch1 | 6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
| | Sample_label_protocol_ch1 | Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
| | Sample_label_protocol_ch1 | IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
| | Sample_label_protocol_ch1 | 1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
| | Sample_label_protocol_ch1 | 2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
| | Sample_label_protocol_ch1 | IVT Cleanup
| | Sample_label_protocol_ch1 | -The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
| | Sample_label_protocol_ch1 | -The RNA elution step is performed twice to increase recovery.
| | Sample_label_protocol_ch1 | -Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
| | Sample_label_protocol_ch1 | Quantifying the cRNA
| | Sample_label_protocol_ch1 | 1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
| | Sample_label_protocol_ch1 | 2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
| | Sample_label_protocol_ch1 | 3.The following formula is used to determine the cRNA yield.
| | Sample_label_protocol_ch1 | Adjusted cRNA yield=RNA* - (total RNA#)($)
| | Sample_label_protocol_ch1 = RNA* | amount of cRNA measured after IVT (mg)
| | Sample_label_protocol_ch1 = total RNA# | sterting amount of total RNA (mg)
| | Sample_label_protocol_ch1 = $ | fraction of cRNA reaction used in IVT
| | Sample_label_protocol_ch1 | cRNA Fragmentation
| | Sample_label_protocol_ch1 | 1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
| | Sample_label_protocol_ch1 | 2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
| | Sample_label_protocol_ch1 | 3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
| | Sample_label_protocol_ch1 | Gel Electrophoresis
| | Sample_label_protocol_ch1 | 1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
| | Sample_label_protocol_ch1 | 2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
| | Sample_hyb_protocol | Hybridization
| | Sample_hyb_protocol | 1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
| | Sample_hyb_protocol | 2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
| | Sample_hyb_protocol | 3.Prepare the hybridization cocktail as follows:
| | Sample_hyb_protocol | Reagents Mini Array Midi Array Standard Array
| | Sample_hyb_protocol | Fragmented cRNA 5mg 10mg 15 mg
| | Sample_hyb_protocol | Control oligo B2 1.7ml 3.3ml 5ml
| | Sample_hyb_protocol | 20X eukaryotic hybridization controls 5ml 10ml 15ml
| | Sample_hyb_protocol | Herring sperm DNA 1ml 2ml 3ml
| | Sample_hyb_protocol | Acetylated BSA 1ml 2ml 3ml
| | Sample_hyb_protocol | 2X Hybridization buffer 50ml 100ml 150mul
| | Sample_hyb_protocol | Water to 100ml to 200ml to 300ml
| | Sample_hyb_protocol |
| | Sample_hyb_protocol | 4.Equilibrate probe array to room temperature.
| | Sample_hyb_protocol | 5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
| | Sample_hyb_protocol | 6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
| | Sample_hyb_protocol | 7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
| | Sample_hyb_protocol | 8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
| | Sample_hyb_protocol | Washing and Staining Probe Arrays
| | Sample_hyb_protocol | Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
| | Sample_scan_protocol | The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
| | Sample_data_processing | dChip (version 1.3) with log2 scaled. Normalized.
| | Sample_platform_id | GPL1261
| | Sample_contact_name | Rui,,Zhu
| | Sample_contact_email | rzhu@mdanderson.org
| | Sample_contact_laboratory | Matin
| | Sample_contact_department | Genetics
| | Sample_contact_institute | UT MD Anderson Cancer Center
| | Sample_contact_address | 1515 Holcombe Blvd.
| | Sample_contact_city | Houston
| | Sample_contact_state | TX
| | Sample_contact_zip/postal_code | 77030
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM357nnn/GSM357103/suppl/GSM357103.CEL.gz
| | Sample_series_id | GSE14481
| | Sample_data_row_count | 45101
| | |
|
GSM357104 | GPL1261 |
|
E13.5 Mouse testis_129/Sv_2
|
E13.5 male genital ridges 129/Sv
|
Background Srain: 129/SV
Gender: male
Tissue: testis
Age: E13.5
|
E13.5 Mouse testis_129/Sv_2
|
| Sample_geo_accession | GSM357104
| | Sample_status | Public on Dec 31 2009
| | Sample_submission_date | Jan 02 2009
| | Sample_last_update_date | Jan 21 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Mus musculus
| | Sample_taxid_ch1 | 10090
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | 40 pairs of testes were pooled for two affymetrix arrays. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
| | Sample_label_ch1 | Biotin
| | Sample_label_protocol_ch1 | Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
| | Sample_label_protocol_ch1 | Use a starting amount of 5.0 – 40.0 mg total RNA.
| | Sample_label_protocol_ch1 | First Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
| | Sample_label_protocol_ch1 | 2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | 3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
| | Sample_label_protocol_ch1 | 4.Incubate at 42° C for 2 minutes.
| | Sample_label_protocol_ch1 | 5.Add to the tube 1 mL of Superscript II reverse transcriptase.
| | Sample_label_protocol_ch1 | 6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
| | Sample_label_protocol_ch1 | 2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
| | Sample_label_protocol_ch1 | 3.Add 2 mL [10 U] T4 DNA Polymerase.
| | Sample_label_protocol_ch1 | 4.Incubate at 16° C for 5 minutes.
| | Sample_label_protocol_ch1 | 5.Add 10 mL 0.5 M EDTA.
| | Sample_label_protocol_ch1 | Cleanup of Double-Stranded cDNA
| | Sample_label_protocol_ch1 | Phenol/Chloroform Extraction
| | Sample_label_protocol_ch1 | 1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
| | Sample_label_protocol_ch1 | 2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
| | Sample_label_protocol_ch1 | Ethanol Precipitation
| | Sample_label_protocol_ch1 | 1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
| | Sample_label_protocol_ch1 | 2.Immediately spin at maximum speed for 20 minutes at room temperature.
| | Sample_label_protocol_ch1 | 3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 5.Air dry pellet.
| | Sample_label_protocol_ch1 | 6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
| | Sample_label_protocol_ch1 | Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
| | Sample_label_protocol_ch1 | IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
| | Sample_label_protocol_ch1 | 1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
| | Sample_label_protocol_ch1 | 2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
| | Sample_label_protocol_ch1 | IVT Cleanup
| | Sample_label_protocol_ch1 | -The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
| | Sample_label_protocol_ch1 | -The RNA elution step is performed twice to increase recovery.
| | Sample_label_protocol_ch1 | -Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
| | Sample_label_protocol_ch1 | Quantifying the cRNA
| | Sample_label_protocol_ch1 | 1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
| | Sample_label_protocol_ch1 | 2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
| | Sample_label_protocol_ch1 | 3.The following formula is used to determine the cRNA yield.
| | Sample_label_protocol_ch1 | Adjusted cRNA yield=RNA* - (total RNA#)($)
| | Sample_label_protocol_ch1 = RNA* | amount of cRNA measured after IVT (mg)
| | Sample_label_protocol_ch1 = total RNA# | sterting amount of total RNA (mg)
| | Sample_label_protocol_ch1 = $ | fraction of cRNA reaction used in IVT
| | Sample_label_protocol_ch1 | cRNA Fragmentation
| | Sample_label_protocol_ch1 | 1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
| | Sample_label_protocol_ch1 | 2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
| | Sample_label_protocol_ch1 | 3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
| | Sample_label_protocol_ch1 | Gel Electrophoresis
| | Sample_label_protocol_ch1 | 1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
| | Sample_label_protocol_ch1 | 2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
| | Sample_hyb_protocol | Hybridization
| | Sample_hyb_protocol | 1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
| | Sample_hyb_protocol | 2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
| | Sample_hyb_protocol | 3.Prepare the hybridization cocktail as follows:
| | Sample_hyb_protocol | Reagents Mini Array Midi Array Standard Array
| | Sample_hyb_protocol | Fragmented cRNA 5mg 10mg 15 mg
| | Sample_hyb_protocol | Control oligo B2 1.7ml 3.3ml 5ml
| | Sample_hyb_protocol | 20X eukaryotic hybridization controls 5ml 10ml 15ml
| | Sample_hyb_protocol | Herring sperm DNA 1ml 2ml 3ml
| | Sample_hyb_protocol | Acetylated BSA 1ml 2ml 3ml
| | Sample_hyb_protocol | 2X Hybridization buffer 50ml 100ml 150mul
| | Sample_hyb_protocol | Water to 100ml to 200ml to 300ml
| | Sample_hyb_protocol |
| | Sample_hyb_protocol | 4.Equilibrate probe array to room temperature.
| | Sample_hyb_protocol | 5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
| | Sample_hyb_protocol | 6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
| | Sample_hyb_protocol | 7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
| | Sample_hyb_protocol | 8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
| | Sample_hyb_protocol | Washing and Staining Probe Arrays
| | Sample_hyb_protocol | Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
| | Sample_scan_protocol | The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
| | Sample_data_processing | dChip (version 1.3) with log2 scaled. Normalized.
| | Sample_platform_id | GPL1261
| | Sample_contact_name | Rui,,Zhu
| | Sample_contact_email | rzhu@mdanderson.org
| | Sample_contact_laboratory | Matin
| | Sample_contact_department | Genetics
| | Sample_contact_institute | UT MD Anderson Cancer Center
| | Sample_contact_address | 1515 Holcombe Blvd.
| | Sample_contact_city | Houston
| | Sample_contact_state | TX
| | Sample_contact_zip/postal_code | 77030
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM357nnn/GSM357104/suppl/GSM357104.CEL.gz
| | Sample_series_id | GSE14481
| | Sample_data_row_count | 45101
| | |
|
GSM357105 | GPL1261 |
|
E13.5 Mouse testis_M19_1
|
E13.5 male genital ridges, 129.MOLF-Chr 19
|
Background Srain: 129.MOLF-Chr 19
Gender: male
Tissue: testis
Age: E13.5
|
E13.5 Mouse testis_M19_1
|
| Sample_geo_accession | GSM357105
| | Sample_status | Public on Dec 31 2009
| | Sample_submission_date | Jan 02 2009
| | Sample_last_update_date | Jan 21 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Mus musculus
| | Sample_taxid_ch1 | 10090
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | 40 pairs of testes were pooled for two affymetrix arrays. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
| | Sample_label_ch1 | Biotin
| | Sample_label_protocol_ch1 | Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
| | Sample_label_protocol_ch1 | Use a starting amount of 5.0 – 40.0 mg total RNA.
| | Sample_label_protocol_ch1 | First Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
| | Sample_label_protocol_ch1 | 2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | 3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
| | Sample_label_protocol_ch1 | 4.Incubate at 42° C for 2 minutes.
| | Sample_label_protocol_ch1 | 5.Add to the tube 1 mL of Superscript II reverse transcriptase.
| | Sample_label_protocol_ch1 | 6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
| | Sample_label_protocol_ch1 | 2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
| | Sample_label_protocol_ch1 | 3.Add 2 mL [10 U] T4 DNA Polymerase.
| | Sample_label_protocol_ch1 | 4.Incubate at 16° C for 5 minutes.
| | Sample_label_protocol_ch1 | 5.Add 10 mL 0.5 M EDTA.
| | Sample_label_protocol_ch1 | Cleanup of Double-Stranded cDNA
| | Sample_label_protocol_ch1 | Phenol/Chloroform Extraction
| | Sample_label_protocol_ch1 | 1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
| | Sample_label_protocol_ch1 | 2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
| | Sample_label_protocol_ch1 | Ethanol Precipitation
| | Sample_label_protocol_ch1 | 1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
| | Sample_label_protocol_ch1 | 2.Immediately spin at maximum speed for 20 minutes at room temperature.
| | Sample_label_protocol_ch1 | 3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 5.Air dry pellet.
| | Sample_label_protocol_ch1 | 6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
| | Sample_label_protocol_ch1 | Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
| | Sample_label_protocol_ch1 | IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
| | Sample_label_protocol_ch1 | 1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
| | Sample_label_protocol_ch1 | 2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
| | Sample_label_protocol_ch1 | IVT Cleanup
| | Sample_label_protocol_ch1 | -The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
| | Sample_label_protocol_ch1 | -The RNA elution step is performed twice to increase recovery.
| | Sample_label_protocol_ch1 | -Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
| | Sample_label_protocol_ch1 | Quantifying the cRNA
| | Sample_label_protocol_ch1 | 1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
| | Sample_label_protocol_ch1 | 2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
| | Sample_label_protocol_ch1 | 3.The following formula is used to determine the cRNA yield.
| | Sample_label_protocol_ch1 | Adjusted cRNA yield=RNA* - (total RNA#)($)
| | Sample_label_protocol_ch1 = RNA* | amount of cRNA measured after IVT (mg)
| | Sample_label_protocol_ch1 = total RNA# | sterting amount of total RNA (mg)
| | Sample_label_protocol_ch1 = $ | fraction of cRNA reaction used in IVT
| | Sample_label_protocol_ch1 | cRNA Fragmentation
| | Sample_label_protocol_ch1 | 1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
| | Sample_label_protocol_ch1 | 2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
| | Sample_label_protocol_ch1 | 3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
| | Sample_label_protocol_ch1 | Gel Electrophoresis
| | Sample_label_protocol_ch1 | 1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
| | Sample_label_protocol_ch1 | 2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
| | Sample_hyb_protocol | Hybridization
| | Sample_hyb_protocol | 1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
| | Sample_hyb_protocol | 2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
| | Sample_hyb_protocol | 3.Prepare the hybridization cocktail as follows:
| | Sample_hyb_protocol | Reagents Mini Array Midi Array Standard Array
| | Sample_hyb_protocol | Fragmented cRNA 5mg 10mg 15 mg
| | Sample_hyb_protocol | Control oligo B2 1.7ml 3.3ml 5ml
| | Sample_hyb_protocol | 20X eukaryotic hybridization controls 5ml 10ml 15ml
| | Sample_hyb_protocol | Herring sperm DNA 1ml 2ml 3ml
| | Sample_hyb_protocol | Acetylated BSA 1ml 2ml 3ml
| | Sample_hyb_protocol | 2X Hybridization buffer 50ml 100ml 150mul
| | Sample_hyb_protocol | Water to 100ml to 200ml to 300ml
| | Sample_hyb_protocol |
| | Sample_hyb_protocol | 4.Equilibrate probe array to room temperature.
| | Sample_hyb_protocol | 5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
| | Sample_hyb_protocol | 6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
| | Sample_hyb_protocol | 7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
| | Sample_hyb_protocol | 8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
| | Sample_hyb_protocol | Washing and Staining Probe Arrays
| | Sample_hyb_protocol | Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
| | Sample_scan_protocol | The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
| | Sample_data_processing | dChip (version 1.3) with log2 scaled. Normalized.
| | Sample_platform_id | GPL1261
| | Sample_contact_name | Rui,,Zhu
| | Sample_contact_email | rzhu@mdanderson.org
| | Sample_contact_laboratory | Matin
| | Sample_contact_department | Genetics
| | Sample_contact_institute | UT MD Anderson Cancer Center
| | Sample_contact_address | 1515 Holcombe Blvd.
| | Sample_contact_city | Houston
| | Sample_contact_state | TX
| | Sample_contact_zip/postal_code | 77030
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM357nnn/GSM357105/suppl/GSM357105.CEL.gz
| | Sample_series_id | GSE14481
| | Sample_data_row_count | 45101
| | |
|
GSM357106 | GPL1261 |
|
E13.5 Mouse testis_M19_2
|
E13.5 male genital ridges, 129.MOLF-Chr 19
|
Background Srain: 129.MOLF-Chr 19
Gender: male
Tissue: testis
Age: E13.5
|
E13.5 Mouse testis_M19_2
|
| Sample_geo_accession | GSM357106
| | Sample_status | Public on Dec 31 2009
| | Sample_submission_date | Jan 02 2009
| | Sample_last_update_date | Jan 21 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Mus musculus
| | Sample_taxid_ch1 | 10090
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | 40 pairs of testes were pooled for two affymetrix arrays. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
| | Sample_label_ch1 | Biotin
| | Sample_label_protocol_ch1 | Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
| | Sample_label_protocol_ch1 | Use a starting amount of 5.0 – 40.0 mg total RNA.
| | Sample_label_protocol_ch1 | First Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
| | Sample_label_protocol_ch1 | 2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | 3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
| | Sample_label_protocol_ch1 | 4.Incubate at 42° C for 2 minutes.
| | Sample_label_protocol_ch1 | 5.Add to the tube 1 mL of Superscript II reverse transcriptase.
| | Sample_label_protocol_ch1 | 6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
| | Sample_label_protocol_ch1 | 2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
| | Sample_label_protocol_ch1 | 3.Add 2 mL [10 U] T4 DNA Polymerase.
| | Sample_label_protocol_ch1 | 4.Incubate at 16° C for 5 minutes.
| | Sample_label_protocol_ch1 | 5.Add 10 mL 0.5 M EDTA.
| | Sample_label_protocol_ch1 | Cleanup of Double-Stranded cDNA
| | Sample_label_protocol_ch1 | Phenol/Chloroform Extraction
| | Sample_label_protocol_ch1 | 1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
| | Sample_label_protocol_ch1 | 2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
| | Sample_label_protocol_ch1 | Ethanol Precipitation
| | Sample_label_protocol_ch1 | 1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
| | Sample_label_protocol_ch1 | 2.Immediately spin at maximum speed for 20 minutes at room temperature.
| | Sample_label_protocol_ch1 | 3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 5.Air dry pellet.
| | Sample_label_protocol_ch1 | 6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
| | Sample_label_protocol_ch1 | Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
| | Sample_label_protocol_ch1 | IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
| | Sample_label_protocol_ch1 | 1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
| | Sample_label_protocol_ch1 | 2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
| | Sample_label_protocol_ch1 | IVT Cleanup
| | Sample_label_protocol_ch1 | -The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
| | Sample_label_protocol_ch1 | -The RNA elution step is performed twice to increase recovery.
| | Sample_label_protocol_ch1 | -Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
| | Sample_label_protocol_ch1 | Quantifying the cRNA
| | Sample_label_protocol_ch1 | 1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
| | Sample_label_protocol_ch1 | 2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
| | Sample_label_protocol_ch1 | 3.The following formula is used to determine the cRNA yield.
| | Sample_label_protocol_ch1 | Adjusted cRNA yield=RNA* - (total RNA#)($)
| | Sample_label_protocol_ch1 = RNA* | amount of cRNA measured after IVT (mg)
| | Sample_label_protocol_ch1 = total RNA# | sterting amount of total RNA (mg)
| | Sample_label_protocol_ch1 = $ | fraction of cRNA reaction used in IVT
| | Sample_label_protocol_ch1 | cRNA Fragmentation
| | Sample_label_protocol_ch1 | 1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
| | Sample_label_protocol_ch1 | 2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
| | Sample_label_protocol_ch1 | 3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
| | Sample_label_protocol_ch1 | Gel Electrophoresis
| | Sample_label_protocol_ch1 | 1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
| | Sample_label_protocol_ch1 | 2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
| | Sample_hyb_protocol | Hybridization
| | Sample_hyb_protocol | 1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
| | Sample_hyb_protocol | 2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
| | Sample_hyb_protocol | 3.Prepare the hybridization cocktail as follows:
| | Sample_hyb_protocol | Reagents Mini Array Midi Array Standard Array
| | Sample_hyb_protocol | Fragmented cRNA 5mg 10mg 15 mg
| | Sample_hyb_protocol | Control oligo B2 1.7ml 3.3ml 5ml
| | Sample_hyb_protocol | 20X eukaryotic hybridization controls 5ml 10ml 15ml
| | Sample_hyb_protocol | Herring sperm DNA 1ml 2ml 3ml
| | Sample_hyb_protocol | Acetylated BSA 1ml 2ml 3ml
| | Sample_hyb_protocol | 2X Hybridization buffer 50ml 100ml 150mul
| | Sample_hyb_protocol | Water to 100ml to 200ml to 300ml
| | Sample_hyb_protocol |
| | Sample_hyb_protocol | 4.Equilibrate probe array to room temperature.
| | Sample_hyb_protocol | 5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
| | Sample_hyb_protocol | 6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
| | Sample_hyb_protocol | 7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
| | Sample_hyb_protocol | 8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
| | Sample_hyb_protocol | Washing and Staining Probe Arrays
| | Sample_hyb_protocol | Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
| | Sample_scan_protocol | The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
| | Sample_data_processing | dChip (version 1.3) with log2 scaled. Normalized.
| | Sample_platform_id | GPL1261
| | Sample_contact_name | Rui,,Zhu
| | Sample_contact_email | rzhu@mdanderson.org
| | Sample_contact_laboratory | Matin
| | Sample_contact_department | Genetics
| | Sample_contact_institute | UT MD Anderson Cancer Center
| | Sample_contact_address | 1515 Holcombe Blvd.
| | Sample_contact_city | Houston
| | Sample_contact_state | TX
| | Sample_contact_zip/postal_code | 77030
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM357nnn/GSM357106/suppl/GSM357106.CEL.gz
| | Sample_series_id | GSE14481
| | Sample_data_row_count | 45101
| | |
|
GSM357107 | GPL1261 |
|
E15.5 Mouse testis_129/Sv_1
|
E15.5 male genital ridges, 129/Sv
|
Background Srain: 129/Sv
Gender: male
Tissue: testis
Age: E15.5
|
E15.5 Mouse testis_129/Sv_1
|
| Sample_geo_accession | GSM357107
| | Sample_status | Public on Dec 31 2009
| | Sample_submission_date | Jan 02 2009
| | Sample_last_update_date | Jan 21 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Mus musculus
| | Sample_taxid_ch1 | 10090
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | 40 pairs of testes were pooled for two affymetrix arrays. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
| | Sample_label_ch1 | Biotin
| | Sample_label_protocol_ch1 | Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
| | Sample_label_protocol_ch1 | Use a starting amount of 5.0 – 40.0 mg total RNA.
| | Sample_label_protocol_ch1 | First Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
| | Sample_label_protocol_ch1 | 2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | 3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
| | Sample_label_protocol_ch1 | 4.Incubate at 42° C for 2 minutes.
| | Sample_label_protocol_ch1 | 5.Add to the tube 1 mL of Superscript II reverse transcriptase.
| | Sample_label_protocol_ch1 | 6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
| | Sample_label_protocol_ch1 | 2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
| | Sample_label_protocol_ch1 | 3.Add 2 mL [10 U] T4 DNA Polymerase.
| | Sample_label_protocol_ch1 | 4.Incubate at 16° C for 5 minutes.
| | Sample_label_protocol_ch1 | 5.Add 10 mL 0.5 M EDTA.
| | Sample_label_protocol_ch1 | Cleanup of Double-Stranded cDNA
| | Sample_label_protocol_ch1 | Phenol/Chloroform Extraction
| | Sample_label_protocol_ch1 | 1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
| | Sample_label_protocol_ch1 | 2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
| | Sample_label_protocol_ch1 | Ethanol Precipitation
| | Sample_label_protocol_ch1 | 1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
| | Sample_label_protocol_ch1 | 2.Immediately spin at maximum speed for 20 minutes at room temperature.
| | Sample_label_protocol_ch1 | 3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 5.Air dry pellet.
| | Sample_label_protocol_ch1 | 6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
| | Sample_label_protocol_ch1 | Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
| | Sample_label_protocol_ch1 | IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
| | Sample_label_protocol_ch1 | 1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
| | Sample_label_protocol_ch1 | 2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
| | Sample_label_protocol_ch1 | IVT Cleanup
| | Sample_label_protocol_ch1 | -The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
| | Sample_label_protocol_ch1 | -The RNA elution step is performed twice to increase recovery.
| | Sample_label_protocol_ch1 | -Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
| | Sample_label_protocol_ch1 | Quantifying the cRNA
| | Sample_label_protocol_ch1 | 1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
| | Sample_label_protocol_ch1 | 2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
| | Sample_label_protocol_ch1 | 3.The following formula is used to determine the cRNA yield.
| | Sample_label_protocol_ch1 | Adjusted cRNA yield=RNA* - (total RNA#)($)
| | Sample_label_protocol_ch1 = RNA* | amount of cRNA measured after IVT (mg)
| | Sample_label_protocol_ch1 = total RNA# | sterting amount of total RNA (mg)
| | Sample_label_protocol_ch1 = $ | fraction of cRNA reaction used in IVT
| | Sample_label_protocol_ch1 | cRNA Fragmentation
| | Sample_label_protocol_ch1 | 1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
| | Sample_label_protocol_ch1 | 2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
| | Sample_label_protocol_ch1 | 3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
| | Sample_label_protocol_ch1 | Gel Electrophoresis
| | Sample_label_protocol_ch1 | 1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
| | Sample_label_protocol_ch1 | 2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
| | Sample_hyb_protocol | Hybridization
| | Sample_hyb_protocol | 1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
| | Sample_hyb_protocol | 2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
| | Sample_hyb_protocol | 3.Prepare the hybridization cocktail as follows:
| | Sample_hyb_protocol | Reagents Mini Array Midi Array Standard Array
| | Sample_hyb_protocol | Fragmented cRNA 5mg 10mg 15 mg
| | Sample_hyb_protocol | Control oligo B2 1.7ml 3.3ml 5ml
| | Sample_hyb_protocol | 20X eukaryotic hybridization controls 5ml 10ml 15ml
| | Sample_hyb_protocol | Herring sperm DNA 1ml 2ml 3ml
| | Sample_hyb_protocol | Acetylated BSA 1ml 2ml 3ml
| | Sample_hyb_protocol | 2X Hybridization buffer 50ml 100ml 150mul
| | Sample_hyb_protocol | Water to 100ml to 200ml to 300ml
| | Sample_hyb_protocol |
| | Sample_hyb_protocol | 4.Equilibrate probe array to room temperature.
| | Sample_hyb_protocol | 5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
| | Sample_hyb_protocol | 6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
| | Sample_hyb_protocol | 7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
| | Sample_hyb_protocol | 8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
| | Sample_hyb_protocol | Washing and Staining Probe Arrays
| | Sample_hyb_protocol | Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
| | Sample_scan_protocol | The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
| | Sample_data_processing | dChip (version 1.3) with log2 scaled. Normalized.
| | Sample_platform_id | GPL1261
| | Sample_contact_name | Rui,,Zhu
| | Sample_contact_email | rzhu@mdanderson.org
| | Sample_contact_laboratory | Matin
| | Sample_contact_department | Genetics
| | Sample_contact_institute | UT MD Anderson Cancer Center
| | Sample_contact_address | 1515 Holcombe Blvd.
| | Sample_contact_city | Houston
| | Sample_contact_state | TX
| | Sample_contact_zip/postal_code | 77030
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM357nnn/GSM357107/suppl/GSM357107.CEL.gz
| | Sample_series_id | GSE14481
| | Sample_data_row_count | 45101
| | |
|
GSM357108 | GPL1261 |
|
E15.5 Mouse testis_129/Sv_2
|
E15.5 male genital ridges, 129/Sv
|
Background Srain: 129/Sv
Gender: male
Tissue: testis
Age: E15.5
|
E15.5 Mouse testis_129/Sv_2
|
| Sample_geo_accession | GSM357108
| | Sample_status | Public on Dec 31 2009
| | Sample_submission_date | Jan 02 2009
| | Sample_last_update_date | Jan 21 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Mus musculus
| | Sample_taxid_ch1 | 10090
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | 40 pairs of testes were pooled for two affymetrix arrays. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
| | Sample_label_ch1 | Biotin
| | Sample_label_protocol_ch1 | Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
| | Sample_label_protocol_ch1 | Use a starting amount of 5.0 – 40.0 mg total RNA.
| | Sample_label_protocol_ch1 | First Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
| | Sample_label_protocol_ch1 | 2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | 3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
| | Sample_label_protocol_ch1 | 4.Incubate at 42° C for 2 minutes.
| | Sample_label_protocol_ch1 | 5.Add to the tube 1 mL of Superscript II reverse transcriptase.
| | Sample_label_protocol_ch1 | 6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
| | Sample_label_protocol_ch1 | 2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
| | Sample_label_protocol_ch1 | 3.Add 2 mL [10 U] T4 DNA Polymerase.
| | Sample_label_protocol_ch1 | 4.Incubate at 16° C for 5 minutes.
| | Sample_label_protocol_ch1 | 5.Add 10 mL 0.5 M EDTA.
| | Sample_label_protocol_ch1 | Cleanup of Double-Stranded cDNA
| | Sample_label_protocol_ch1 | Phenol/Chloroform Extraction
| | Sample_label_protocol_ch1 | 1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
| | Sample_label_protocol_ch1 | 2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
| | Sample_label_protocol_ch1 | Ethanol Precipitation
| | Sample_label_protocol_ch1 | 1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
| | Sample_label_protocol_ch1 | 2.Immediately spin at maximum speed for 20 minutes at room temperature.
| | Sample_label_protocol_ch1 | 3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 5.Air dry pellet.
| | Sample_label_protocol_ch1 | 6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
| | Sample_label_protocol_ch1 | Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
| | Sample_label_protocol_ch1 | IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
| | Sample_label_protocol_ch1 | 1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
| | Sample_label_protocol_ch1 | 2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
| | Sample_label_protocol_ch1 | IVT Cleanup
| | Sample_label_protocol_ch1 | -The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
| | Sample_label_protocol_ch1 | -The RNA elution step is performed twice to increase recovery.
| | Sample_label_protocol_ch1 | -Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
| | Sample_label_protocol_ch1 | Quantifying the cRNA
| | Sample_label_protocol_ch1 | 1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
| | Sample_label_protocol_ch1 | 2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
| | Sample_label_protocol_ch1 | 3.The following formula is used to determine the cRNA yield.
| | Sample_label_protocol_ch1 | Adjusted cRNA yield=RNA* - (total RNA#)($)
| | Sample_label_protocol_ch1 = RNA* | amount of cRNA measured after IVT (mg)
| | Sample_label_protocol_ch1 = total RNA# | sterting amount of total RNA (mg)
| | Sample_label_protocol_ch1 = $ | fraction of cRNA reaction used in IVT
| | Sample_label_protocol_ch1 | cRNA Fragmentation
| | Sample_label_protocol_ch1 | 1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
| | Sample_label_protocol_ch1 | 2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
| | Sample_label_protocol_ch1 | 3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
| | Sample_label_protocol_ch1 | Gel Electrophoresis
| | Sample_label_protocol_ch1 | 1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
| | Sample_label_protocol_ch1 | 2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
| | Sample_hyb_protocol | Hybridization
| | Sample_hyb_protocol | 1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
| | Sample_hyb_protocol | 2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
| | Sample_hyb_protocol | 3.Prepare the hybridization cocktail as follows:
| | Sample_hyb_protocol | Reagents Mini Array Midi Array Standard Array
| | Sample_hyb_protocol | Fragmented cRNA 5mg 10mg 15 mg
| | Sample_hyb_protocol | Control oligo B2 1.7ml 3.3ml 5ml
| | Sample_hyb_protocol | 20X eukaryotic hybridization controls 5ml 10ml 15ml
| | Sample_hyb_protocol | Herring sperm DNA 1ml 2ml 3ml
| | Sample_hyb_protocol | Acetylated BSA 1ml 2ml 3ml
| | Sample_hyb_protocol | 2X Hybridization buffer 50ml 100ml 150mul
| | Sample_hyb_protocol | Water to 100ml to 200ml to 300ml
| | Sample_hyb_protocol |
| | Sample_hyb_protocol | 4.Equilibrate probe array to room temperature.
| | Sample_hyb_protocol | 5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
| | Sample_hyb_protocol | 6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
| | Sample_hyb_protocol | 7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
| | Sample_hyb_protocol | 8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
| | Sample_hyb_protocol | Washing and Staining Probe Arrays
| | Sample_hyb_protocol | Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
| | Sample_scan_protocol | The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
| | Sample_data_processing | dChip (version 1.3) with log2 scaled. Normalized.
| | Sample_platform_id | GPL1261
| | Sample_contact_name | Rui,,Zhu
| | Sample_contact_email | rzhu@mdanderson.org
| | Sample_contact_laboratory | Matin
| | Sample_contact_department | Genetics
| | Sample_contact_institute | UT MD Anderson Cancer Center
| | Sample_contact_address | 1515 Holcombe Blvd.
| | Sample_contact_city | Houston
| | Sample_contact_state | TX
| | Sample_contact_zip/postal_code | 77030
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM357nnn/GSM357108/suppl/GSM357108.CEL.gz
| | Sample_series_id | GSE14481
| | Sample_data_row_count | 45101
| | |
|
GSM357109 | GPL1261 |
|
E15.5 Mouse testis_M19_1
|
E15.5 male genital ridges, 129.MOLF-Chr 19
|
Background Srain: 129.MOLF-Chr 19
Gender: male
Tissue: testis
Age: E15.5
|
E15.5 Mouse testis_M19_1
|
| Sample_geo_accession | GSM357109
| | Sample_status | Public on Dec 31 2009
| | Sample_submission_date | Jan 02 2009
| | Sample_last_update_date | Jan 21 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Mus musculus
| | Sample_taxid_ch1 | 10090
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | 40 pairs of testes were pooled for two affymetrix arrays. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
| | Sample_label_ch1 | Biotin
| | Sample_label_protocol_ch1 | Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
| | Sample_label_protocol_ch1 | Use a starting amount of 5.0 – 40.0 mg total RNA.
| | Sample_label_protocol_ch1 | First Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
| | Sample_label_protocol_ch1 | 2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | 3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
| | Sample_label_protocol_ch1 | 4.Incubate at 42° C for 2 minutes.
| | Sample_label_protocol_ch1 | 5.Add to the tube 1 mL of Superscript II reverse transcriptase.
| | Sample_label_protocol_ch1 | 6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
| | Sample_label_protocol_ch1 | 2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
| | Sample_label_protocol_ch1 | 3.Add 2 mL [10 U] T4 DNA Polymerase.
| | Sample_label_protocol_ch1 | 4.Incubate at 16° C for 5 minutes.
| | Sample_label_protocol_ch1 | 5.Add 10 mL 0.5 M EDTA.
| | Sample_label_protocol_ch1 | Cleanup of Double-Stranded cDNA
| | Sample_label_protocol_ch1 | Phenol/Chloroform Extraction
| | Sample_label_protocol_ch1 | 1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
| | Sample_label_protocol_ch1 | 2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
| | Sample_label_protocol_ch1 | Ethanol Precipitation
| | Sample_label_protocol_ch1 | 1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
| | Sample_label_protocol_ch1 | 2.Immediately spin at maximum speed for 20 minutes at room temperature.
| | Sample_label_protocol_ch1 | 3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 5.Air dry pellet.
| | Sample_label_protocol_ch1 | 6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
| | Sample_label_protocol_ch1 | Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
| | Sample_label_protocol_ch1 | IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
| | Sample_label_protocol_ch1 | 1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
| | Sample_label_protocol_ch1 | 2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
| | Sample_label_protocol_ch1 | IVT Cleanup
| | Sample_label_protocol_ch1 | -The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
| | Sample_label_protocol_ch1 | -The RNA elution step is performed twice to increase recovery.
| | Sample_label_protocol_ch1 | -Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
| | Sample_label_protocol_ch1 | Quantifying the cRNA
| | Sample_label_protocol_ch1 | 1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
| | Sample_label_protocol_ch1 | 2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
| | Sample_label_protocol_ch1 | 3.The following formula is used to determine the cRNA yield.
| | Sample_label_protocol_ch1 | Adjusted cRNA yield=RNA* - (total RNA#)($)
| | Sample_label_protocol_ch1 = RNA* | amount of cRNA measured after IVT (mg)
| | Sample_label_protocol_ch1 = total RNA# | sterting amount of total RNA (mg)
| | Sample_label_protocol_ch1 = $ | fraction of cRNA reaction used in IVT
| | Sample_label_protocol_ch1 | cRNA Fragmentation
| | Sample_label_protocol_ch1 | 1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
| | Sample_label_protocol_ch1 | 2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
| | Sample_label_protocol_ch1 | 3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
| | Sample_label_protocol_ch1 | Gel Electrophoresis
| | Sample_label_protocol_ch1 | 1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
| | Sample_label_protocol_ch1 | 2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
| | Sample_hyb_protocol | Hybridization
| | Sample_hyb_protocol | 1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
| | Sample_hyb_protocol | 2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
| | Sample_hyb_protocol | 3.Prepare the hybridization cocktail as follows:
| | Sample_hyb_protocol | Reagents Mini Array Midi Array Standard Array
| | Sample_hyb_protocol | Fragmented cRNA 5mg 10mg 15 mg
| | Sample_hyb_protocol | Control oligo B2 1.7ml 3.3ml 5ml
| | Sample_hyb_protocol | 20X eukaryotic hybridization controls 5ml 10ml 15ml
| | Sample_hyb_protocol | Herring sperm DNA 1ml 2ml 3ml
| | Sample_hyb_protocol | Acetylated BSA 1ml 2ml 3ml
| | Sample_hyb_protocol | 2X Hybridization buffer 50ml 100ml 150mul
| | Sample_hyb_protocol | Water to 100ml to 200ml to 300ml
| | Sample_hyb_protocol |
| | Sample_hyb_protocol | 4.Equilibrate probe array to room temperature.
| | Sample_hyb_protocol | 5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
| | Sample_hyb_protocol | 6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
| | Sample_hyb_protocol | 7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
| | Sample_hyb_protocol | 8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
| | Sample_hyb_protocol | Washing and Staining Probe Arrays
| | Sample_hyb_protocol | Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
| | Sample_scan_protocol | The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
| | Sample_data_processing | dChip (version 1.3) with log2 scaled. Normalized.
| | Sample_platform_id | GPL1261
| | Sample_contact_name | Rui,,Zhu
| | Sample_contact_email | rzhu@mdanderson.org
| | Sample_contact_laboratory | Matin
| | Sample_contact_department | Genetics
| | Sample_contact_institute | UT MD Anderson Cancer Center
| | Sample_contact_address | 1515 Holcombe Blvd.
| | Sample_contact_city | Houston
| | Sample_contact_state | TX
| | Sample_contact_zip/postal_code | 77030
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM357nnn/GSM357109/suppl/GSM357109.CEL.gz
| | Sample_series_id | GSE14481
| | Sample_data_row_count | 45101
| | |
|
GSM357110 | GPL1261 |
|
E15.5 Mouse testis_M19_2
|
E15.5 male genital ridges, 129.MOLF-Chr 19
|
Background Srain: 129.MOLF-Chr 19
Gender: male
Tissue: testis
Age: E15.5
|
E15.5 Mouse testis_M19_2
|
| Sample_geo_accession | GSM357110
| | Sample_status | Public on Dec 31 2009
| | Sample_submission_date | Jan 02 2009
| | Sample_last_update_date | Jan 21 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Mus musculus
| | Sample_taxid_ch1 | 10090
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | 40 pairs of testes were pooled for two affymetrix arrays. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
| | Sample_label_ch1 | Biotin
| | Sample_label_protocol_ch1 | Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
| | Sample_label_protocol_ch1 | Use a starting amount of 5.0 – 40.0 mg total RNA.
| | Sample_label_protocol_ch1 | First Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
| | Sample_label_protocol_ch1 | 2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | 3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
| | Sample_label_protocol_ch1 | 4.Incubate at 42° C for 2 minutes.
| | Sample_label_protocol_ch1 | 5.Add to the tube 1 mL of Superscript II reverse transcriptase.
| | Sample_label_protocol_ch1 | 6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
| | Sample_label_protocol_ch1 | 2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
| | Sample_label_protocol_ch1 | 3.Add 2 mL [10 U] T4 DNA Polymerase.
| | Sample_label_protocol_ch1 | 4.Incubate at 16° C for 5 minutes.
| | Sample_label_protocol_ch1 | 5.Add 10 mL 0.5 M EDTA.
| | Sample_label_protocol_ch1 | Cleanup of Double-Stranded cDNA
| | Sample_label_protocol_ch1 | Phenol/Chloroform Extraction
| | Sample_label_protocol_ch1 | 1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
| | Sample_label_protocol_ch1 | 2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
| | Sample_label_protocol_ch1 | Ethanol Precipitation
| | Sample_label_protocol_ch1 | 1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
| | Sample_label_protocol_ch1 | 2.Immediately spin at maximum speed for 20 minutes at room temperature.
| | Sample_label_protocol_ch1 | 3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 5.Air dry pellet.
| | Sample_label_protocol_ch1 | 6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
| | Sample_label_protocol_ch1 | Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
| | Sample_label_protocol_ch1 | IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
| | Sample_label_protocol_ch1 | 1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
| | Sample_label_protocol_ch1 | 2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
| | Sample_label_protocol_ch1 | IVT Cleanup
| | Sample_label_protocol_ch1 | -The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
| | Sample_label_protocol_ch1 | -The RNA elution step is performed twice to increase recovery.
| | Sample_label_protocol_ch1 | -Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
| | Sample_label_protocol_ch1 | Quantifying the cRNA
| | Sample_label_protocol_ch1 | 1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
| | Sample_label_protocol_ch1 | 2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
| | Sample_label_protocol_ch1 | 3.The following formula is used to determine the cRNA yield.
| | Sample_label_protocol_ch1 | Adjusted cRNA yield=RNA* - (total RNA#)($)
| | Sample_label_protocol_ch1 = RNA* | amount of cRNA measured after IVT (mg)
| | Sample_label_protocol_ch1 = total RNA# | sterting amount of total RNA (mg)
| | Sample_label_protocol_ch1 = $ | fraction of cRNA reaction used in IVT
| | Sample_label_protocol_ch1 | cRNA Fragmentation
| | Sample_label_protocol_ch1 | 1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
| | Sample_label_protocol_ch1 | 2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
| | Sample_label_protocol_ch1 | 3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
| | Sample_label_protocol_ch1 | Gel Electrophoresis
| | Sample_label_protocol_ch1 | 1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
| | Sample_label_protocol_ch1 | 2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
| | Sample_hyb_protocol | Hybridization
| | Sample_hyb_protocol | 1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
| | Sample_hyb_protocol | 2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
| | Sample_hyb_protocol | 3.Prepare the hybridization cocktail as follows:
| | Sample_hyb_protocol | Reagents Mini Array Midi Array Standard Array
| | Sample_hyb_protocol | Fragmented cRNA 5mg 10mg 15 mg
| | Sample_hyb_protocol | Control oligo B2 1.7ml 3.3ml 5ml
| | Sample_hyb_protocol | 20X eukaryotic hybridization controls 5ml 10ml 15ml
| | Sample_hyb_protocol | Herring sperm DNA 1ml 2ml 3ml
| | Sample_hyb_protocol | Acetylated BSA 1ml 2ml 3ml
| | Sample_hyb_protocol | 2X Hybridization buffer 50ml 100ml 150mul
| | Sample_hyb_protocol | Water to 100ml to 200ml to 300ml
| | Sample_hyb_protocol |
| | Sample_hyb_protocol | 4.Equilibrate probe array to room temperature.
| | Sample_hyb_protocol | 5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
| | Sample_hyb_protocol | 6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
| | Sample_hyb_protocol | 7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
| | Sample_hyb_protocol | 8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
| | Sample_hyb_protocol | Washing and Staining Probe Arrays
| | Sample_hyb_protocol | Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
| | Sample_scan_protocol | The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
| | Sample_data_processing | dChip (version 1.3) with log2 scaled. Normalized.
| | Sample_platform_id | GPL1261
| | Sample_contact_name | Rui,,Zhu
| | Sample_contact_email | rzhu@mdanderson.org
| | Sample_contact_laboratory | Matin
| | Sample_contact_department | Genetics
| | Sample_contact_institute | UT MD Anderson Cancer Center
| | Sample_contact_address | 1515 Holcombe Blvd.
| | Sample_contact_city | Houston
| | Sample_contact_state | TX
| | Sample_contact_zip/postal_code | 77030
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM357nnn/GSM357110/suppl/GSM357110.CEL.gz
| | Sample_series_id | GSE14481
| | Sample_data_row_count | 45101
| | |
|
GSM357111 | GPL1261 |
|
E17.5 Mouse testis_129/Sv_1
|
E17.5 male genital ridges, 129/Sv
|
Background Srain: 129/Sv
Gender: male
Tissue: testis
Age: E17.5
|
E17.5 Mouse testis_129/Sv_1
|
| Sample_geo_accession | GSM357111
| | Sample_status | Public on Dec 31 2009
| | Sample_submission_date | Jan 02 2009
| | Sample_last_update_date | Jan 21 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Mus musculus
| | Sample_taxid_ch1 | 10090
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | 40 pairs of testes were pooled for two affymetrix arrays. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
| | Sample_label_ch1 | Biotin
| | Sample_label_protocol_ch1 | Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
| | Sample_label_protocol_ch1 | Use a starting amount of 5.0 – 40.0 mg total RNA.
| | Sample_label_protocol_ch1 | First Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
| | Sample_label_protocol_ch1 | 2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | 3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
| | Sample_label_protocol_ch1 | 4.Incubate at 42° C for 2 minutes.
| | Sample_label_protocol_ch1 | 5.Add to the tube 1 mL of Superscript II reverse transcriptase.
| | Sample_label_protocol_ch1 | 6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
| | Sample_label_protocol_ch1 | 2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
| | Sample_label_protocol_ch1 | 3.Add 2 mL [10 U] T4 DNA Polymerase.
| | Sample_label_protocol_ch1 | 4.Incubate at 16° C for 5 minutes.
| | Sample_label_protocol_ch1 | 5.Add 10 mL 0.5 M EDTA.
| | Sample_label_protocol_ch1 | Cleanup of Double-Stranded cDNA
| | Sample_label_protocol_ch1 | Phenol/Chloroform Extraction
| | Sample_label_protocol_ch1 | 1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
| | Sample_label_protocol_ch1 | 2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
| | Sample_label_protocol_ch1 | Ethanol Precipitation
| | Sample_label_protocol_ch1 | 1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
| | Sample_label_protocol_ch1 | 2.Immediately spin at maximum speed for 20 minutes at room temperature.
| | Sample_label_protocol_ch1 | 3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 5.Air dry pellet.
| | Sample_label_protocol_ch1 | 6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
| | Sample_label_protocol_ch1 | Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
| | Sample_label_protocol_ch1 | IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
| | Sample_label_protocol_ch1 | 1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
| | Sample_label_protocol_ch1 | 2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
| | Sample_label_protocol_ch1 | IVT Cleanup
| | Sample_label_protocol_ch1 | -The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
| | Sample_label_protocol_ch1 | -The RNA elution step is performed twice to increase recovery.
| | Sample_label_protocol_ch1 | -Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
| | Sample_label_protocol_ch1 | Quantifying the cRNA
| | Sample_label_protocol_ch1 | 1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
| | Sample_label_protocol_ch1 | 2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
| | Sample_label_protocol_ch1 | 3.The following formula is used to determine the cRNA yield.
| | Sample_label_protocol_ch1 | Adjusted cRNA yield=RNA* - (total RNA#)($)
| | Sample_label_protocol_ch1 = RNA* | amount of cRNA measured after IVT (mg)
| | Sample_label_protocol_ch1 = total RNA# | sterting amount of total RNA (mg)
| | Sample_label_protocol_ch1 = $ | fraction of cRNA reaction used in IVT
| | Sample_label_protocol_ch1 | cRNA Fragmentation
| | Sample_label_protocol_ch1 | 1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
| | Sample_label_protocol_ch1 | 2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
| | Sample_label_protocol_ch1 | 3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
| | Sample_label_protocol_ch1 | Gel Electrophoresis
| | Sample_label_protocol_ch1 | 1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
| | Sample_label_protocol_ch1 | 2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
| | Sample_hyb_protocol | Hybridization
| | Sample_hyb_protocol | 1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
| | Sample_hyb_protocol | 2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
| | Sample_hyb_protocol | 3.Prepare the hybridization cocktail as follows:
| | Sample_hyb_protocol | Reagents Mini Array Midi Array Standard Array
| | Sample_hyb_protocol | Fragmented cRNA 5mg 10mg 15 mg
| | Sample_hyb_protocol | Control oligo B2 1.7ml 3.3ml 5ml
| | Sample_hyb_protocol | 20X eukaryotic hybridization controls 5ml 10ml 15ml
| | Sample_hyb_protocol | Herring sperm DNA 1ml 2ml 3ml
| | Sample_hyb_protocol | Acetylated BSA 1ml 2ml 3ml
| | Sample_hyb_protocol | 2X Hybridization buffer 50ml 100ml 150mul
| | Sample_hyb_protocol | Water to 100ml to 200ml to 300ml
| | Sample_hyb_protocol |
| | Sample_hyb_protocol | 4.Equilibrate probe array to room temperature.
| | Sample_hyb_protocol | 5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
| | Sample_hyb_protocol | 6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
| | Sample_hyb_protocol | 7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
| | Sample_hyb_protocol | 8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
| | Sample_hyb_protocol | Washing and Staining Probe Arrays
| | Sample_hyb_protocol | Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
| | Sample_scan_protocol | The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
| | Sample_data_processing | dChip (version 1.3) with log2 scaled. Normalized.
| | Sample_platform_id | GPL1261
| | Sample_contact_name | Rui,,Zhu
| | Sample_contact_email | rzhu@mdanderson.org
| | Sample_contact_laboratory | Matin
| | Sample_contact_department | Genetics
| | Sample_contact_institute | UT MD Anderson Cancer Center
| | Sample_contact_address | 1515 Holcombe Blvd.
| | Sample_contact_city | Houston
| | Sample_contact_state | TX
| | Sample_contact_zip/postal_code | 77030
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM357nnn/GSM357111/suppl/GSM357111.CEL.gz
| | Sample_series_id | GSE14481
| | Sample_data_row_count | 45101
| | |
|
GSM357112 | GPL1261 |
|
E17.5 Mouse testis_129/Sv_2
|
E17.5 male genital ridges, 129/Sv
|
Background Srain: 129/Sv
Gender: male
Tissue: testis
Age: E17.5
|
E17.5 Mouse testis_129/Sv_2
|
| Sample_geo_accession | GSM357112
| | Sample_status | Public on Dec 31 2009
| | Sample_submission_date | Jan 02 2009
| | Sample_last_update_date | Jan 21 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Mus musculus
| | Sample_taxid_ch1 | 10090
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | 40 pairs of testes were pooled for two affymetrix arrays. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
| | Sample_label_ch1 | Biotin
| | Sample_label_protocol_ch1 | Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
| | Sample_label_protocol_ch1 | Use a starting amount of 5.0 – 40.0 mg total RNA.
| | Sample_label_protocol_ch1 | First Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
| | Sample_label_protocol_ch1 | 2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | 3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
| | Sample_label_protocol_ch1 | 4.Incubate at 42° C for 2 minutes.
| | Sample_label_protocol_ch1 | 5.Add to the tube 1 mL of Superscript II reverse transcriptase.
| | Sample_label_protocol_ch1 | 6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
| | Sample_label_protocol_ch1 | 2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
| | Sample_label_protocol_ch1 | 3.Add 2 mL [10 U] T4 DNA Polymerase.
| | Sample_label_protocol_ch1 | 4.Incubate at 16° C for 5 minutes.
| | Sample_label_protocol_ch1 | 5.Add 10 mL 0.5 M EDTA.
| | Sample_label_protocol_ch1 | Cleanup of Double-Stranded cDNA
| | Sample_label_protocol_ch1 | Phenol/Chloroform Extraction
| | Sample_label_protocol_ch1 | 1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
| | Sample_label_protocol_ch1 | 2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
| | Sample_label_protocol_ch1 | Ethanol Precipitation
| | Sample_label_protocol_ch1 | 1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
| | Sample_label_protocol_ch1 | 2.Immediately spin at maximum speed for 20 minutes at room temperature.
| | Sample_label_protocol_ch1 | 3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 5.Air dry pellet.
| | Sample_label_protocol_ch1 | 6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
| | Sample_label_protocol_ch1 | Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
| | Sample_label_protocol_ch1 | IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
| | Sample_label_protocol_ch1 | 1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
| | Sample_label_protocol_ch1 | 2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
| | Sample_label_protocol_ch1 | IVT Cleanup
| | Sample_label_protocol_ch1 | -The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
| | Sample_label_protocol_ch1 | -The RNA elution step is performed twice to increase recovery.
| | Sample_label_protocol_ch1 | -Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
| | Sample_label_protocol_ch1 | Quantifying the cRNA
| | Sample_label_protocol_ch1 | 1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
| | Sample_label_protocol_ch1 | 2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
| | Sample_label_protocol_ch1 | 3.The following formula is used to determine the cRNA yield.
| | Sample_label_protocol_ch1 | Adjusted cRNA yield=RNA* - (total RNA#)($)
| | Sample_label_protocol_ch1 = RNA* | amount of cRNA measured after IVT (mg)
| | Sample_label_protocol_ch1 = total RNA# | sterting amount of total RNA (mg)
| | Sample_label_protocol_ch1 = $ | fraction of cRNA reaction used in IVT
| | Sample_label_protocol_ch1 | cRNA Fragmentation
| | Sample_label_protocol_ch1 | 1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
| | Sample_label_protocol_ch1 | 2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
| | Sample_label_protocol_ch1 | 3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
| | Sample_label_protocol_ch1 | Gel Electrophoresis
| | Sample_label_protocol_ch1 | 1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
| | Sample_label_protocol_ch1 | 2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
| | Sample_hyb_protocol | Hybridization
| | Sample_hyb_protocol | 1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
| | Sample_hyb_protocol | 2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
| | Sample_hyb_protocol | 3.Prepare the hybridization cocktail as follows:
| | Sample_hyb_protocol | Reagents Mini Array Midi Array Standard Array
| | Sample_hyb_protocol | Fragmented cRNA 5mg 10mg 15 mg
| | Sample_hyb_protocol | Control oligo B2 1.7ml 3.3ml 5ml
| | Sample_hyb_protocol | 20X eukaryotic hybridization controls 5ml 10ml 15ml
| | Sample_hyb_protocol | Herring sperm DNA 1ml 2ml 3ml
| | Sample_hyb_protocol | Acetylated BSA 1ml 2ml 3ml
| | Sample_hyb_protocol | 2X Hybridization buffer 50ml 100ml 150mul
| | Sample_hyb_protocol | Water to 100ml to 200ml to 300ml
| | Sample_hyb_protocol |
| | Sample_hyb_protocol | 4.Equilibrate probe array to room temperature.
| | Sample_hyb_protocol | 5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
| | Sample_hyb_protocol | 6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
| | Sample_hyb_protocol | 7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
| | Sample_hyb_protocol | 8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
| | Sample_hyb_protocol | Washing and Staining Probe Arrays
| | Sample_hyb_protocol | Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
| | Sample_scan_protocol | The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
| | Sample_data_processing | dChip (version 1.3) with log2 scaled. Normalized.
| | Sample_platform_id | GPL1261
| | Sample_contact_name | Rui,,Zhu
| | Sample_contact_email | rzhu@mdanderson.org
| | Sample_contact_laboratory | Matin
| | Sample_contact_department | Genetics
| | Sample_contact_institute | UT MD Anderson Cancer Center
| | Sample_contact_address | 1515 Holcombe Blvd.
| | Sample_contact_city | Houston
| | Sample_contact_state | TX
| | Sample_contact_zip/postal_code | 77030
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM357nnn/GSM357112/suppl/GSM357112.CEL.gz
| | Sample_series_id | GSE14481
| | Sample_data_row_count | 45101
| | |
|
GSM357113 | GPL1261 |
|
E17.5 Mouse testis_M19_1
|
E17.5 male genital ridges, 129.MOLF-Chr 19
|
Background Srain: 129.MOLF-Chr 19
Gender: male
Tissue: testis
Age: E17.5
|
E17.5 Mouse testis_M19_1
|
| Sample_geo_accession | GSM357113
| | Sample_status | Public on Dec 31 2009
| | Sample_submission_date | Jan 02 2009
| | Sample_last_update_date | Jan 21 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Mus musculus
| | Sample_taxid_ch1 | 10090
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | 40 pairs of testes were pooled for two affymetrix arrays. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
| | Sample_label_ch1 | Biotin
| | Sample_label_protocol_ch1 | Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
| | Sample_label_protocol_ch1 | Use a starting amount of 5.0 – 40.0 mg total RNA.
| | Sample_label_protocol_ch1 | First Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
| | Sample_label_protocol_ch1 | 2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | 3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
| | Sample_label_protocol_ch1 | 4.Incubate at 42° C for 2 minutes.
| | Sample_label_protocol_ch1 | 5.Add to the tube 1 mL of Superscript II reverse transcriptase.
| | Sample_label_protocol_ch1 | 6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
| | Sample_label_protocol_ch1 | 2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
| | Sample_label_protocol_ch1 | 3.Add 2 mL [10 U] T4 DNA Polymerase.
| | Sample_label_protocol_ch1 | 4.Incubate at 16° C for 5 minutes.
| | Sample_label_protocol_ch1 | 5.Add 10 mL 0.5 M EDTA.
| | Sample_label_protocol_ch1 | Cleanup of Double-Stranded cDNA
| | Sample_label_protocol_ch1 | Phenol/Chloroform Extraction
| | Sample_label_protocol_ch1 | 1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
| | Sample_label_protocol_ch1 | 2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
| | Sample_label_protocol_ch1 | Ethanol Precipitation
| | Sample_label_protocol_ch1 | 1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
| | Sample_label_protocol_ch1 | 2.Immediately spin at maximum speed for 20 minutes at room temperature.
| | Sample_label_protocol_ch1 | 3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 5.Air dry pellet.
| | Sample_label_protocol_ch1 | 6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
| | Sample_label_protocol_ch1 | Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
| | Sample_label_protocol_ch1 | IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
| | Sample_label_protocol_ch1 | 1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
| | Sample_label_protocol_ch1 | 2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
| | Sample_label_protocol_ch1 | IVT Cleanup
| | Sample_label_protocol_ch1 | -The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
| | Sample_label_protocol_ch1 | -The RNA elution step is performed twice to increase recovery.
| | Sample_label_protocol_ch1 | -Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
| | Sample_label_protocol_ch1 | Quantifying the cRNA
| | Sample_label_protocol_ch1 | 1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
| | Sample_label_protocol_ch1 | 2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
| | Sample_label_protocol_ch1 | 3.The following formula is used to determine the cRNA yield.
| | Sample_label_protocol_ch1 | Adjusted cRNA yield=RNA* - (total RNA#)($)
| | Sample_label_protocol_ch1 = RNA* | amount of cRNA measured after IVT (mg)
| | Sample_label_protocol_ch1 = total RNA# | sterting amount of total RNA (mg)
| | Sample_label_protocol_ch1 = $ | fraction of cRNA reaction used in IVT
| | Sample_label_protocol_ch1 | cRNA Fragmentation
| | Sample_label_protocol_ch1 | 1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
| | Sample_label_protocol_ch1 | 2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
| | Sample_label_protocol_ch1 | 3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
| | Sample_label_protocol_ch1 | Gel Electrophoresis
| | Sample_label_protocol_ch1 | 1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
| | Sample_label_protocol_ch1 | 2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
| | Sample_hyb_protocol | Hybridization
| | Sample_hyb_protocol | 1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
| | Sample_hyb_protocol | 2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
| | Sample_hyb_protocol | 3.Prepare the hybridization cocktail as follows:
| | Sample_hyb_protocol | Reagents Mini Array Midi Array Standard Array
| | Sample_hyb_protocol | Fragmented cRNA 5mg 10mg 15 mg
| | Sample_hyb_protocol | Control oligo B2 1.7ml 3.3ml 5ml
| | Sample_hyb_protocol | 20X eukaryotic hybridization controls 5ml 10ml 15ml
| | Sample_hyb_protocol | Herring sperm DNA 1ml 2ml 3ml
| | Sample_hyb_protocol | Acetylated BSA 1ml 2ml 3ml
| | Sample_hyb_protocol | 2X Hybridization buffer 50ml 100ml 150mul
| | Sample_hyb_protocol | Water to 100ml to 200ml to 300ml
| | Sample_hyb_protocol |
| | Sample_hyb_protocol | 4.Equilibrate probe array to room temperature.
| | Sample_hyb_protocol | 5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
| | Sample_hyb_protocol | 6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
| | Sample_hyb_protocol | 7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
| | Sample_hyb_protocol | 8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
| | Sample_hyb_protocol | Washing and Staining Probe Arrays
| | Sample_hyb_protocol | Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
| | Sample_scan_protocol | The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
| | Sample_data_processing | dChip (version 1.3) with log2 scaled. Normalized.
| | Sample_platform_id | GPL1261
| | Sample_contact_name | Rui,,Zhu
| | Sample_contact_email | rzhu@mdanderson.org
| | Sample_contact_laboratory | Matin
| | Sample_contact_department | Genetics
| | Sample_contact_institute | UT MD Anderson Cancer Center
| | Sample_contact_address | 1515 Holcombe Blvd.
| | Sample_contact_city | Houston
| | Sample_contact_state | TX
| | Sample_contact_zip/postal_code | 77030
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM357nnn/GSM357113/suppl/GSM357113.CEL.gz
| | Sample_series_id | GSE14481
| | Sample_data_row_count | 45101
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GSM357114 | GPL1261 |
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E17.5 Mouse testis_M19_2
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E17.5 male genital ridges, 129.MOLF-Chr 19
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Background Srain: 129.MOLF-Chr 19
Gender: male
Tissue: testis
Age: E17.5
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E17.5 Mouse testis_M19_2
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| Sample_geo_accession | GSM357114
| | Sample_status | Public on Dec 31 2009
| | Sample_submission_date | Jan 02 2009
| | Sample_last_update_date | Jan 21 2009
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Mus musculus
| | Sample_taxid_ch1 | 10090
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | 40 pairs of testes were pooled for two affymetrix arrays. RNA was isolated using Qiagen RNeasy mini kits. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.
| | Sample_label_ch1 | Biotin
| | Sample_label_protocol_ch1 | Synthesis of Double Stranded cDNA from Total RNA (Using the Supercript Choice System by Gibco)
| | Sample_label_protocol_ch1 | Use a starting amount of 5.0 – 40.0 mg total RNA.
| | Sample_label_protocol_ch1 | First Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.In a 1.5 mL microcentrifuge tube, combine the following: x mL of total RNA, 12- x mL of RNase/DNase free water and 1 mL of T7- (dT)24 primer.
| | Sample_label_protocol_ch1 | 2.Incubate at 70° C for 10 minutes. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | 3.Add the following to the tube: 4 mL of 5X First strand cDNA buffer, 2 mL of 0.1 M DTT, and 1 mL of 10 mM dNTP mix.
| | Sample_label_protocol_ch1 | 4.Incubate at 42° C for 2 minutes.
| | Sample_label_protocol_ch1 | 5.Add to the tube 1 mL of Superscript II reverse transcriptase.
| | Sample_label_protocol_ch1 | 6.Incubate at 42° C for 1 hour. Centrifuge to collect contents at the bottom of the tube. Put on ice.
| | Sample_label_protocol_ch1 | Second Strand cDNA Synthesis (using 5.0 mg of total RNA)
| | Sample_label_protocol_ch1 | 1.Add the following to the First Strand Synthesis tube: 91 mL of RNase/DNase free water, 30 mL of 5X Second strand reaction buffer, 3 mL of 10 mM dNTP mix, 1 mL of 10 U/mL DNA Ligase (E. coli), 4 mL of 10 U/mL DNA Polymerase I (E. coli), and 1 mL of 2 U/mL RNase H (E. coli).
| | Sample_label_protocol_ch1 | 2.Tap tube to mix. Centrifuge to collect contents at the bottom of the tube. Incubate at 16°C for 2 hours.
| | Sample_label_protocol_ch1 | 3.Add 2 mL [10 U] T4 DNA Polymerase.
| | Sample_label_protocol_ch1 | 4.Incubate at 16° C for 5 minutes.
| | Sample_label_protocol_ch1 | 5.Add 10 mL 0.5 M EDTA.
| | Sample_label_protocol_ch1 | Cleanup of Double-Stranded cDNA
| | Sample_label_protocol_ch1 | Phenol/Chloroform Extraction
| | Sample_label_protocol_ch1 | 1.Add 162 mL of (25:24:1) Phenol:chloroform:isoamyl alcohol. Vortex.
| | Sample_label_protocol_ch1 | 2.Centrifuge sample at maximum speed for 2 minutes.Transfer aqueous phase to fresh 1.5 mL microcentrifuge tube.
| | Sample_label_protocol_ch1 | Ethanol Precipitation
| | Sample_label_protocol_ch1 | 1.Add 10 mg/mL of glycogen, 0.5 volumes of 7.5 M ammonium acetate, and 2.5 volumes of absolute ethanol to sample. Vortex.
| | Sample_label_protocol_ch1 | 2.Immediately spin at maximum speed for 20 minutes at room temperature.
| | Sample_label_protocol_ch1 | 3.Remove supernatant. Wash pellet with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 4.Remove 80% ethanol. Wash again with 500 mL of 80% ethanol. Spin at maximum speed for 5 minutes at room temperature.
| | Sample_label_protocol_ch1 | 5.Air dry pellet.
| | Sample_label_protocol_ch1 | 6.Resuspend in 12 mL of RNase/Dnase free water. (You can store at -20°C. Save 1-2 mL aliquot to run on gel later.
| | Sample_label_protocol_ch1 | Synthesis of Biotin-Labeled cRNA (Using In Vitro Transcription Reaction)
| | Sample_label_protocol_ch1 | IVT Reaction (Using the ENZO BioArray High Yield RNA Transcript Labeling Kit)
| | Sample_label_protocol_ch1 | 1.Add the following to a fresh 1.5 mL microcentrifuge tube: x mL to give 1 mg double-stranded cDNA, 22 – x mL of RNase/Dnase free water, 4 mL of 10 X HY reaction buffer, 4 mL of 10 X Biotin labeled ribonucleotides, 4 mL of 10 X DTT, 4 mL of 10 X RNase inhibitor mix, and 2 mL of T7 RNA polymerase.
| | Sample_label_protocol_ch1 | 2.Incubate at 37°C for 4 – 5 hours, mixing tube every 30 – 45 minutes. (You can store at -20°C until ready to purify.
| | Sample_label_protocol_ch1 | IVT Cleanup
| | Sample_label_protocol_ch1 | -The preferred method for cRNA cleanup is by using RNeasy spin columns from Qiagen.
| | Sample_label_protocol_ch1 | -The RNA elution step is performed twice to increase recovery.
| | Sample_label_protocol_ch1 | -Ethanol precipitation (performed as described above) is performed following RNA elution and the RNA pellet is resuspended in 18 ml of water.
| | Sample_label_protocol_ch1 | Quantifying the cRNA
| | Sample_label_protocol_ch1 | 1.Prepare 1:50 dilution of the RNA to a total of 100 ml.
| | Sample_label_protocol_ch1 | 2.Check OD at 260nm and 280 nm to determine sample concentration and purity. A 260/A280 ratio between 1.9 and 2.1 is acceptable.
| | Sample_label_protocol_ch1 | 3.The following formula is used to determine the cRNA yield.
| | Sample_label_protocol_ch1 | Adjusted cRNA yield=RNA* - (total RNA#)($)
| | Sample_label_protocol_ch1 = RNA* | amount of cRNA measured after IVT (mg)
| | Sample_label_protocol_ch1 = total RNA# | sterting amount of total RNA (mg)
| | Sample_label_protocol_ch1 = $ | fraction of cRNA reaction used in IVT
| | Sample_label_protocol_ch1 | cRNA Fragmentation
| | Sample_label_protocol_ch1 | 1.To 20 mg of unadjusted cRNA 2 ml of 5X fragmentation buffer is added for every 8 ml of RNA and the total volume is adjusted to 40ml with water.
| | Sample_label_protocol_ch1 | 2.The fragmentation reaction is performed at 94C for 35 minutes and kept on ice post incubation.
| | Sample_label_protocol_ch1 | 3.An aliquot of cRNA is saved for gel analysis and the rest is stored at –20C until ready to perform hybridization.
| | Sample_label_protocol_ch1 | Gel Electrophoresis
| | Sample_label_protocol_ch1 | 1.Run aliquots of purified cDNA (after ethanol precipitation), purified cRNA and fragmented cRNA on 1% agarose gel.
| | Sample_label_protocol_ch1 | 2.Mix RNA with loading dye and heat to 65C for 5 minutes before loading on the gel.
| | Sample_hyb_protocol | Hybridization
| | Sample_hyb_protocol | 1.Thaw 1 vial of the 20X eukaryotic hybridization controls and the vial of oligo B2 (included in the GeneChip Eukaryotic hybridization control kit) by heating the vials at 65C for 5 minutes.
| | Sample_hyb_protocol | 2.Vortex the vials to ensure complete mixing and then briefly spin down the samples.
| | Sample_hyb_protocol | 3.Prepare the hybridization cocktail as follows:
| | Sample_hyb_protocol | Reagents Mini Array Midi Array Standard Array
| | Sample_hyb_protocol | Fragmented cRNA 5mg 10mg 15 mg
| | Sample_hyb_protocol | Control oligo B2 1.7ml 3.3ml 5ml
| | Sample_hyb_protocol | 20X eukaryotic hybridization controls 5ml 10ml 15ml
| | Sample_hyb_protocol | Herring sperm DNA 1ml 2ml 3ml
| | Sample_hyb_protocol | Acetylated BSA 1ml 2ml 3ml
| | Sample_hyb_protocol | 2X Hybridization buffer 50ml 100ml 150mul
| | Sample_hyb_protocol | Water to 100ml to 200ml to 300ml
| | Sample_hyb_protocol |
| | Sample_hyb_protocol | 4.Equilibrate probe array to room temperature.
| | Sample_hyb_protocol | 5.Heat the hybridization cocktail to 99C for 5 minutes, spin briefly and transfer to 45C for 5 minutes.
| | Sample_hyb_protocol | 6.Spin hybridization/cocktail at maximum speed for 5 minutes to remove any insoluble material.
| | Sample_hyb_protocol | 7.Wet the probe array by filling it through one of the septa with 1X hybridization buffer and incubate at 45C for 10 minutes with rotation.
| | Sample_hyb_protocol | 8.Remove the buffer solution from the probe array and fill the probe array with the appropriate amount of the clarified hybridization cocktail. Hybridize overnight at 45C with rotation.
| | Sample_hyb_protocol | Washing and Staining Probe Arrays
| | Sample_hyb_protocol | Follow instructions for operation and usage of fluidics station as given in the Expression Analysis technical manual from Affymetrix.
| | Sample_scan_protocol | The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center.
| | Sample_data_processing | dChip (version 1.3) with log2 scaled. Normalized.
| | Sample_platform_id | GPL1261
| | Sample_contact_name | Rui,,Zhu
| | Sample_contact_email | rzhu@mdanderson.org
| | Sample_contact_laboratory | Matin
| | Sample_contact_department | Genetics
| | Sample_contact_institute | UT MD Anderson Cancer Center
| | Sample_contact_address | 1515 Holcombe Blvd.
| | Sample_contact_city | Houston
| | Sample_contact_state | TX
| | Sample_contact_zip/postal_code | 77030
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM357nnn/GSM357114/suppl/GSM357114.CEL.gz
| | Sample_series_id | GSE14481
| | Sample_data_row_count | 45101
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(2 groups will be compared at a time) |
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| Select GSMs and click on "Add groups" |
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