Search results for the GEO ID: GSE7584  |
(Click on the check boxes provided under "Select for analysis", to initiate grouping) |
(Once the selection is made, click on "Add groups" in "Make groups for comparison", to make a group. Scroll down) |
|
GSM ID | GPL ID |
Select for analysis |
Title |
Source name |
Description |
Characteristics |
GSM183317 | GPL1355 |
|
KSU Dr.Lillich - IEC6 cells treated with NSAIDS - C1
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells
Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings:
Scaling - All Probe Sets: Target Signal = 500
Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183317
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with DMSO for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | First Cycle of Amplification
| | Sample_label_protocol_ch1 | Step 1. First cycle, first strand cDNA synthesis
| | Sample_label_protocol_ch1 | Note: Use C holdthermal cycler with a heated lid for incubations in this step. Use the 4 C 6 minutesfor the addition of reagents. 70
| | Sample_label_protocol_ch1 | C hold4
| | Sample_label_protocol_ch1 | C 6042 minutes
| | Sample_label_protocol_ch1 | C 10 minutes70
| | Sample_label_protocol_ch1 | C hold4
| | Sample_label_protocol_ch1 | a. Mix 1ul of total RNA (50ng/ul) C in thermalwith 1ul of 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min.
| | Sample_label_protocol_ch1 | (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’)
| | Sample_label_protocol_ch1 | b. C for 2 min.Cool to 4
| | Sample_label_protocol_ch1 | c. Spin briefly.
| | Sample_label_protocol_ch1 | d. Add 3ul of RT-Premix-1 master mix using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes.
| | Sample_label_protocol_ch1 | 1 rxn
| | Sample_label_protocol_ch1 | DEPC treated water 0.375ul
| | Sample_label_protocol_ch1 | 5x first strand buffer 1ul
| | Sample_label_protocol_ch1 | DTT, 0.1M 0.5ul
| | Sample_label_protocol_ch1 | dNTP mix, 10mM 0.375ul
| | Sample_label_protocol_ch1 | Rnase inhibitor, 40 U/ul 0.25ul
| | Sample_label_protocol_ch1 | SuperScript II, 200 U/ul 0.5ul
| | Sample_label_protocol_ch1 | Total Volume 3ul
| | Sample_label_protocol_ch1 | C 60 minutes.e. Incubated at 42
| | Sample_label_protocol_ch1 | f. Heat sample C for 10 minutes to inactivate SuperScript II.at 70
| | Sample_label_protocol_ch1 | g. Spin briefly and cool C.sample to 4
| | Sample_label_protocol_ch1 | Step 2. First cycle, second strand cDNA synthesis
| | Sample_label_protocol_ch1 | Note: Use C hold forthermal cycler without heated lid for incubations in this step. Use 4 the addition of reagents.
| | Sample_label_protocol_ch1 | C 120 minutes16
| | Sample_label_protocol_ch1 | C hold4
| | Sample_label_protocol_ch1 | C 1016 minutes
| | Sample_label_protocol_ch1 | C hold4
| | Sample_label_protocol_ch1 | a Prepare the SS-Premix-1 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes.
| | Sample_label_protocol_ch1 | 1 rxn
| | Sample_label_protocol_ch1 | DEPC treated water 22.75ul
| | Sample_label_protocol_ch1 | 5x second strand buffer 7.5ul
| | Sample_label_protocol_ch1 | dNTP mix, 10mM 0.75ul
| | Sample_label_protocol_ch1 | DNA ligase, E. coli, 10U/ul 0.25ul
| | Sample_label_protocol_ch1 | DNA polymerase I, E. coli 10U/ul 1ul
| | Sample_label_protocol_ch1 | Rnase H, 2U/ul 0.25ul
| | Sample_label_protocol_ch1 | Total Volume 32.5ul
| | Sample_label_protocol_ch1 | b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul.
| | Sample_label_protocol_ch1 | c. Mix by pipetting and spin briefly.
| | Sample_label_protocol_ch1 | d. C for 2 hours.Incubate the samples at 16
| | Sample_label_protocol_ch1 | e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting.
| | Sample_label_protocol_ch1 | Cf. Continue incubation at 16 for 10 minutes.
| | Sample_label_protocol_ch1 | Step 3. First cycle, double-stranded cDNA cleanup by ethanol precipitation.
| | Sample_label_protocol_ch1 | a. Transfer the reaction to a 1.5 ml centrifuge tube.
| | Sample_label_protocol_ch1 | b. Add 80ul of DEPC treated water to dilute reaction.
| | Sample_label_protocol_ch1 | c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting.
| | Sample_label_protocol_ch1 | d. Precipitate the double-stranded cDNA C for 2 hours.at -20
| | Sample_label_protocol_ch1 | C.e. Centrifuge at 14,000rpm for 20 minutes at 4
| | Sample_label_protocol_ch1 | f. Wash pellet with 800ul of 70% cold ethanol.
| | Sample_label_protocol_ch1 | g. Centrifuge at 14,000rpm for 5 C.minutes at 4
| | Sample_label_protocol_ch1 | h. Remove ethanol and dry pellet in speed vacuum for 10 minutes.
| | Sample_label_protocol_ch1 | Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion).
| | Sample_label_protocol_ch1 | a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated.
| | Sample_label_protocol_ch1 | 1rxn
| | Sample_label_protocol_ch1 | DEPC treated water 4ul
| | Sample_label_protocol_ch1 | Premixed NTPs, 18.75mM each 4ul
| | Sample_label_protocol_ch1 | 10x reaction buffer 1ul
| | Sample_label_protocol_ch1 | 10x enzyme mix 1ul
| | Sample_label_protocol_ch1 | Total Volume 10ul
| | Sample_label_protocol_ch1 | b. Mix well by pipetting. Spin briefly.
| | Sample_label_protocol_ch1 | C for 5 hours mixing every 30 minutes.c. Incubate at 37
| | Sample_label_protocol_ch1 | Step 5. First cycle, cRNA cleanup with Qiagen RNeasy columns.
| | Sample_label_protocol_ch1 | a. Add 90ul of Rnase-free water to sample.
| | Sample_label_protocol_ch1 | b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook.
| | Sample_label_protocol_ch1 | c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution.
| | Sample_label_protocol_ch1 | d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm.
| | Sample_label_protocol_ch1 | e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis.
| | Sample_label_protocol_ch1 | Second Cycle of Amplification and Labeling
| | Sample_label_protocol_ch1 | Step 6. Second cycle, first strand cDNA synthesis.
| | Sample_label_protocol_ch1 | Note: Use heating blocks for incubation in this step.
| | Sample_label_protocol_ch1 | a. Add random primers to the cRNA sample and mix well.
| | Sample_label_protocol_ch1 | cRNA, 400ng 4ul
| | Sample_label_protocol_ch1 | Random primers, 0.2 ug/ul 1ul
| | Sample_label_protocol_ch1 | Total Volume 5ul
| | Sample_label_protocol_ch1 | C for 10 minutes to denature cRNA.b. Incubate at 70
| | Sample_label_protocol_ch1 | c. Cool sample on ice for 2 minutes.
| | Sample_label_protocol_ch1 | d. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes.
| | Sample_label_protocol_ch1 | 1rxn
| | Sample_label_protocol_ch1 | 5x first strand buffer 2ul
| | Sample_label_protocol_ch1 | DTT, 0.1M 1ul
| | Sample_label_protocol_ch1 | dNTP mix, 10mM 0.5ul
| | Sample_label_protocol_ch1 | RNase Inhibitor, 40 U/ul 0.5ul
| | Sample_label_protocol_ch1 | SuperScript II, 200 U/ul 1ul
| | Sample_label_protocol_ch1 | Total Volume 5ul
| | Sample_label_protocol_ch1 | e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by pipetting. Spin briefly.
| | Sample_label_protocol_ch1 | C for 1 hour. Spin briefly.f. Incubate the sample at 42
| | Sample_label_protocol_ch1 | g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the sample at C for 20 minutes.37
| | Sample_label_protocol_ch1 | C for 5 minutes to inactivateh. Heat the sample at 95 RNase H.
| | Sample_label_protocol_ch1 | i. Cool sample on ice 2 minutes. Spin briefly.
| | Sample_label_protocol_ch1 | Step 7. Second cycle, second strand cDNA synthesis
| | Sample_label_protocol_ch1 | Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold to add reagents.the 4
| | Sample_label_protocol_ch1 | C 6 minutes70
| | Sample_label_protocol_ch1 | C hold4
| | Sample_label_protocol_ch1 | C 12016 minutes
| | Sample_label_protocol_ch1 | C hold4
| | Sample_label_protocol_ch1 | C 10 minutes16
| | Sample_label_protocol_ch1 | C hold4
| | Sample_label_protocol_ch1 | a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the chilled sample from the previous step.
| | Sample_label_protocol_ch1 | b. Mix well. Spin briefly.
| | Sample_label_protocol_ch1 | C for 6 minutes.c. Incubate sample at 70
| | Sample_label_protocol_ch1 | d. Cool the C and spin briefly.sample to 4
| | Sample_label_protocol_ch1 | e. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen).
| | Sample_label_protocol_ch1 | 1rxn
| | Sample_label_protocol_ch1 | DEPC treated water 43.5ul
| | Sample_label_protocol_ch1 | 5x second strand buffer 15ul
| | Sample_label_protocol_ch1 | dNTP mix, 10mM 1.5ul
| | Sample_label_protocol_ch1 | DNA polymerase I, E. coli, 10 U/ul 2ul
| | Sample_label_protocol_ch1 | Total Volume 62ul
| | Sample_label_protocol_ch1 | f. Add 62ul of the SS-Premix-2 to the sample making a total volume of 75ul. Mix well by pipetting. Spin briefly.
| | Sample_label_protocol_ch1 | C for 2 hours.g. Incubate at 16
| | Sample_label_protocol_ch1 | h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well.
| | Sample_label_protocol_ch1 | C for 10i. Incubate at 16 minutes.
| | Sample_label_protocol_ch1 | Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation.
| | Sample_label_protocol_ch1 | a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol.
| | Sample_label_protocol_ch1 | b. Mix well by pipetting.
| | Sample_label_protocol_ch1 | c. C for 30 minutes.Precipitate the double-stranded cDNA at -20
| | Sample_label_protocol_ch1 | d. Centrifuge C.the sample at 14,000 rpm for 20 minutes at 4
| | Sample_label_protocol_ch1 | e. Carefully remove supernatant and wash the cDNA pellet by adding 800ul of 70% cold ethanol.
| | Sample_label_protocol_ch1 | f. C.Centrifuge the tube at 14,000 rpm for 5 minutes at 4
| | Sample_label_protocol_ch1 | g. Carefully remove the ethanol. Dry pellet in a speed vacuum for 10 minutes.
| | Sample_label_protocol_ch1 | h. Store dry pellet C or proceed to next step.at –20
| | Sample_label_protocol_ch1 | Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription
| | Sample_label_protocol_ch1 | Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix).
| | Sample_label_protocol_ch1 | a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet.
| | Sample_label_protocol_ch1 | Enzo BioArray High Yield RNA Transcription Labeling kit
| | Sample_label_protocol_ch1 | 1rxn
| | Sample_label_protocol_ch1 | DEPC treated water 22ul
| | Sample_label_protocol_ch1 | 10X HY reaction buffer 4ul
| | Sample_label_protocol_ch1 | 10X Biotin labeled ribonucleotides 4ul
| | Sample_label_protocol_ch1 | 10X DTT 4ul
| | Sample_label_protocol_ch1 | 10X RNase inhibitor mix 4ul
| | Sample_label_protocol_ch1 | 20X T7 RNA polymerase 2ul
| | Sample_label_protocol_ch1 | Total Volume 40ul
| | Sample_label_protocol_ch1 | 1. Mix the components well after adding each reagent; especially after adding water, ensuring the cDNA
| | Sample_label_protocol_ch1 | pellet is completely dissolved.
| | Sample_label_protocol_ch1 | C for2. Incubate at 37 5 hours mixing every 30 minutes.
| | Sample_label_protocol_ch1 | GeneChip Expression 3’-Amplification IVT Labeling Kit
| | Sample_label_protocol_ch1 | 1rxn
| | Sample_label_protocol_ch1 | DEPC treated water 20ul
| | Sample_label_protocol_ch1 | 10x IVT Labeling Buffer 4ul
| | Sample_label_protocol_ch1 | IVT Labeling NTP Mix 12ul
| | Sample_label_protocol_ch1 | IVT Labeling Enzyme Mix 4ul
| | Sample_label_protocol_ch1 | Total Volume 40ul
| | Sample_label_protocol_ch1 | 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet
| | Sample_label_protocol_ch1 | is completely dissolved.
| | Sample_label_protocol_ch1 | 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent
| | Sample_label_protocol_ch1 | condensation.
| | Sample_label_protocol_ch1 | Step
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL:
| | Sample_hyb_protocol | Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets
| | Sample_hyb_protocol | Prepared using the GeneChip
| | Sample_hyb_protocol | Expression 3’-Amplificaton IVT
| | Sample_hyb_protocol | Labeling Protocol.
| | Sample_hyb_protocol | 30ul fragmented cRNA 15ug (0.05ug/ul final conc.)
| | Sample_hyb_protocol | 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.)
| | Sample_hyb_protocol | 15ul 20x Eukaryotic Hybridization spike controls
| | Sample_hyb_protocol | (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc.
| | Sample_hyb_protocol | respectively)
| | Sample_hyb_protocol | 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.)
| | Sample_hyb_protocol | 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.)
| | Sample_hyb_protocol | 150ul 2x Hybridization Buffer
| | Sample_hyb_protocol | (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES
| | Sample_hyb_protocol | sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20)
| | Sample_hyb_protocol | - DEPC H2O to 300ul.
| | Sample_hyb_protocol | - Denatured at 99C 5min prior to applying to GeneChip
| | Sample_hyb_protocol | Hybridization Conditions:
| | Sample_hyb_protocol | - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm
| | Sample_hyb_protocol | - 200ul denatured Hybridization cocktail is applied to GeneChip
| | Sample_hyb_protocol | - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640.
| | Sample_hyb_protocol | Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard
| | Sample_hyb_protocol | Array Format.
| | Sample_hyb_protocol | Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol
| | Sample_hyb_protocol | Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent
| | Sample_hyb_protocol | (6xSSPE, 0.01% Tween 20)
| | Sample_hyb_protocol | Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM
| | Sample_hyb_protocol | MES, 0.1M [Na+], 0.01% Tween 20)
| | Sample_hyb_protocol | 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES,
| | Sample_hyb_protocol | 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O},
| | Sample_hyb_protocol | 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin
| | Sample_hyb_protocol | conjugate, 270ul DEPC H2O, 600ul total vol.)
| | Sample_hyb_protocol | Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C
| | Sample_hyb_protocol | 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above},
| | Sample_hyb_protocol | 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG,
| | Sample_hyb_protocol | 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O,
| | Sample_hyb_protocol | 600ul total vol.)
| | Sample_hyb_protocol | 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.)
| | Sample_hyb_protocol | Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains
| | Sample_hyb_protocol | filled with wash buffer A for the scanning procedure.
| | Sample_hyb_protocol | GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol
| | Sample_hyb_protocol | Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent
| | Sample_hyb_protocol | (6xSSPE, 0.01% Tween 20)
| | Sample_hyb_protocol | Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM
| | Sample_hyb_protocol | MES, 0.1M [Na+], 0.01% Tween 20)
| | Sample_hyb_protocol | 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES,
| | Sample_hyb_protocol | 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O},
| | Sample_hyb_protocol | 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin
| | Sample_hyb_protocol | conjugate, 270ul DEPC H2O, 600ul total vol.)
| | Sample_hyb_protocol | Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC
| | Sample_hyb_protocol | 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above},
| | Sample_hyb_protocol | 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG,
| | Sample_hyb_protocol | 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O,
| | Sample_hyb_protocol | 600ul total vol.)
| | Sample_hyb_protocol | 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.)
| | Sample_hyb_protocol | Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains
| | Sample_hyb_protocol | filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol | Scanning: Automated process conducted using the Agilent GeneArray Scanner.
| | Sample_scan_protocol | - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip
| | Sample_scan_protocol | Expression protocol as follows:
| | Sample_scan_protocol = Pixel value | 3um
| | Sample_scan_protocol = Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183317/suppl/GSM183317.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183317/suppl/GSM183317.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
GSM183318 | GPL1355 |
|
KSU-Dr.Lillich - IEC6 cells treated with NSAIDS - C2
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells
Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings: Scaling - All Probe Sets: Target Signal = 500 Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183318
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with DMSO for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation.
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL: Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets Prepared using the GeneChip Expression 3’-Amplificaton IVT Labeling Protocol. 30ul fragmented cRNA 15ug (0.05ug/ul final conc.) 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.) 15ul 20x Eukaryotic Hybridization spike controls (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc. respectively) 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.) 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.) 150ul 2x Hybridization Buffer (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20) - DEPC H2O to 300ul. - Denatured at 99C 5min prior to applying to GeneChip Hybridization Conditions: - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm - 200ul denatured Hybridization cocktail is applied to GeneChip - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640. Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard Array Format. Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains filled with wash buffer A for the scanning procedure. GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol = Scanning: Automated process conducted using the Agilent GeneArray Scanner. - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip Expression protocol as follows: Pixel value = 3um Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183318/suppl/GSM183318.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183318/suppl/GSM183318.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
GSM183319 | GPL1355 |
|
KSU Dr.Lillich - IEC6 cells treated with NSAIDS - C1b
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings: Scaling - All Probe Sets: Target Signal = 500 Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183319
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with DMSO for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation.
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL: Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets Prepared using the GeneChip Expression 3’-Amplificaton IVT Labeling Protocol. 30ul fragmented cRNA 15ug (0.05ug/ul final conc.) 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.) 15ul 20x Eukaryotic Hybridization spike controls (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc. respectively) 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.) 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.) 150ul 2x Hybridization Buffer (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20) - DEPC H2O to 300ul. - Denatured at 99C 5min prior to applying to GeneChip Hybridization Conditions: - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm - 200ul denatured Hybridization cocktail is applied to GeneChip - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640. Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard Array Format. Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains filled with wash buffer A for the scanning procedure. GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol = Scanning: Automated process conducted using the Agilent GeneArray Scanner. - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip Expression protocol as follows: Pixel value = 3um Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183319/suppl/GSM183319.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183319/suppl/GSM183319.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
GSM183320 | GPL1355 |
|
KSU Dr.Lillich - IEC6 - cells treated with NSAIDS - C2b
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells
Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings: Scaling - All Probe Sets: Target Signal = 500 Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183320
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with DMSO for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation.
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL: Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets Prepared using the GeneChip Expression 3’-Amplificaton IVT Labeling Protocol. 30ul fragmented cRNA 15ug (0.05ug/ul final conc.) 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.) 15ul 20x Eukaryotic Hybridization spike controls (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc. respectively) 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.) 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.) 150ul 2x Hybridization Buffer (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20) - DEPC H2O to 300ul. - Denatured at 99C 5min prior to applying to GeneChip Hybridization Conditions: - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm - 200ul denatured Hybridization cocktail is applied to GeneChip - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640. Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard Array Format. Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains filled with wash buffer A for the scanning procedure. GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol = Scanning: Automated process conducted using the Agilent GeneArray Scanner. - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip Expression protocol as follows: Pixel value = 3um Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183320/suppl/GSM183320.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183320/suppl/GSM183320.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
GSM183321 | GPL1355 |
|
KSU Dr.Lillich - IEC6 cells treated with NSAIDS - I1b
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells
Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings: Scaling - All Probe Sets: Target Signal = 500 Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183321
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with 100µM Indomethacin for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation.
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL: Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets Prepared using the GeneChip Expression 3’-Amplificaton IVT Labeling Protocol. 30ul fragmented cRNA 15ug (0.05ug/ul final conc.) 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.) 15ul 20x Eukaryotic Hybridization spike controls (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc. respectively) 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.) 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.) 150ul 2x Hybridization Buffer (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20) - DEPC H2O to 300ul. - Denatured at 99C 5min prior to applying to GeneChip Hybridization Conditions: - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm - 200ul denatured Hybridization cocktail is applied to GeneChip - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640. Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard Array Format. Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains filled with wash buffer A for the scanning procedure. GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol = Scanning: Automated process conducted using the Agilent GeneArray Scanner. - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip Expression protocol as follows: Pixel value = 3um Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183321/suppl/GSM183321.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183321/suppl/GSM183321.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
GSM183322 | GPL1355 |
|
KSU Dr.Lillich - IEC6 - cells treated with NSAIDS - I2b
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells
Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings: Scaling - All Probe Sets: Target Signal = 500 Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183322
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with 100µM Indomethacin for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation.
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL: Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets Prepared using the GeneChip Expression 3’-Amplificaton IVT Labeling Protocol. 30ul fragmented cRNA 15ug (0.05ug/ul final conc.) 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.) 15ul 20x Eukaryotic Hybridization spike controls (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc. respectively) 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.) 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.) 150ul 2x Hybridization Buffer (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20) - DEPC H2O to 300ul. - Denatured at 99C 5min prior to applying to GeneChip Hybridization Conditions: - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm - 200ul denatured Hybridization cocktail is applied to GeneChip - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640. Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard Array Format. Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains filled with wash buffer A for the scanning procedure. GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol = Scanning: Automated process conducted using the Agilent GeneArray Scanner. - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip Expression protocol as follows: Pixel value = 3um Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183322/suppl/GSM183322.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183322/suppl/GSM183322.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
GSM183323 | GPL1355 |
|
KSU Dr.Lillich - IEC6 - cells treated with NSAIDS - NS1
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells
Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings: Scaling - All Probe Sets: Target Signal = 500 Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183323
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with 100µM NS-398 for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation.
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL: Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets Prepared using the GeneChip Expression 3’-Amplificaton IVT Labeling Protocol. 30ul fragmented cRNA 15ug (0.05ug/ul final conc.) 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.) 15ul 20x Eukaryotic Hybridization spike controls (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc. respectively) 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.) 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.) 150ul 2x Hybridization Buffer (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20) - DEPC H2O to 300ul. - Denatured at 99C 5min prior to applying to GeneChip Hybridization Conditions: - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm - 200ul denatured Hybridization cocktail is applied to GeneChip - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640. Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard Array Format. Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains filled with wash buffer A for the scanning procedure. GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol = Scanning: Automated process conducted using the Agilent GeneArray Scanner. - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip Expression protocol as follows: Pixel value = 3um Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183323/suppl/GSM183323.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183323/suppl/GSM183323.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
GSM183324 | GPL1355 |
|
KSU Dr.Lillich - IEC6 - cells treated with NSAIDS - NS2
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells
Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings: Scaling - All Probe Sets: Target Signal = 500 Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183324
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with 100µM NS-398 for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation.
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL: Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets Prepared using the GeneChip Expression 3’-Amplificaton IVT Labeling Protocol. 30ul fragmented cRNA 15ug (0.05ug/ul final conc.) 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.) 15ul 20x Eukaryotic Hybridization spike controls (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc. respectively) 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.) 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.) 150ul 2x Hybridization Buffer (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20) - DEPC H2O to 300ul. - Denatured at 99C 5min prior to applying to GeneChip Hybridization Conditions: - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm - 200ul denatured Hybridization cocktail is applied to GeneChip - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640. Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard Array Format. Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains filled with wash buffer A for the scanning procedure. GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol = Scanning: Automated process conducted using the Agilent GeneArray Scanner. - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip Expression protocol as follows: Pixel value = 3um Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183324/suppl/GSM183324.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183324/suppl/GSM183324.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
GSM183325 | GPL1355 |
|
KSU Dr.Lillich - IEC6 - cells treated with NSAIDS - NS1b
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells
Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings: Scaling - All Probe Sets: Target Signal = 500 Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183325
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with 100µM NS-398 for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation.
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL: Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets Prepared using the GeneChip Expression 3’-Amplificaton IVT Labeling Protocol. 30ul fragmented cRNA 15ug (0.05ug/ul final conc.) 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.) 15ul 20x Eukaryotic Hybridization spike controls (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc. respectively) 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.) 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.) 150ul 2x Hybridization Buffer (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20) - DEPC H2O to 300ul. - Denatured at 99C 5min prior to applying to GeneChip Hybridization Conditions: - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm - 200ul denatured Hybridization cocktail is applied to GeneChip - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640. Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard Array Format. Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains filled with wash buffer A for the scanning procedure. GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol = Scanning: Automated process conducted using the Agilent GeneArray Scanner. - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip Expression protocol as follows: Pixel value = 3um Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183325/suppl/GSM183325.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183325/suppl/GSM183325.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
GSM183326 | GPL1355 |
|
KSU Dr.Lillich - IEC6 - cells treated with NSAIDS - NS2b
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells
Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings: Scaling - All Probe Sets: Target Signal = 500 Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183326
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with 100µM NS-398 for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation.
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL: Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets Prepared using the GeneChip Expression 3’-Amplificaton IVT Labeling Protocol. 30ul fragmented cRNA 15ug (0.05ug/ul final conc.) 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.) 15ul 20x Eukaryotic Hybridization spike controls (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc. respectively) 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.) 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.) 150ul 2x Hybridization Buffer (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20) - DEPC H2O to 300ul. - Denatured at 99C 5min prior to applying to GeneChip Hybridization Conditions: - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm - 200ul denatured Hybridization cocktail is applied to GeneChip - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640. Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard Array Format. Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains filled with wash buffer A for the scanning procedure. GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol = Scanning: Automated process conducted using the Agilent GeneArray Scanner. - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip Expression protocol as follows: Pixel value = 3um Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183326/suppl/GSM183326.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183326/suppl/GSM183326.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
GSM183328 | GPL1355 |
|
KSU Dr.Lillich - IEC6 - cells treated with NSAIDS - SC1
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells
Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings: Scaling - All Probe Sets: Target Signal = 500 Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183328
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with 1µM SC-560 for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation.
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL: Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets Prepared using the GeneChip Expression 3’-Amplificaton IVT Labeling Protocol. 30ul fragmented cRNA 15ug (0.05ug/ul final conc.) 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.) 15ul 20x Eukaryotic Hybridization spike controls (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc. respectively) 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.) 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.) 150ul 2x Hybridization Buffer (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20) - DEPC H2O to 300ul. - Denatured at 99C 5min prior to applying to GeneChip Hybridization Conditions: - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm - 200ul denatured Hybridization cocktail is applied to GeneChip - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640. Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard Array Format. Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains filled with wash buffer A for the scanning procedure. GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol = Scanning: Automated process conducted using the Agilent GeneArray Scanner. - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip Expression protocol as follows: Pixel value = 3um Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183328/suppl/GSM183328.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183328/suppl/GSM183328.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
GSM183330 | GPL1355 |
|
KSU Dr.Lillich - IEC6 - cells treated with NSAIDS - SC2
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells
Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings: Scaling - All Probe Sets: Target Signal = 500 Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183330
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with 1µM SC-560 for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation.
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL: Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets Prepared using the GeneChip Expression 3’-Amplificaton IVT Labeling Protocol. 30ul fragmented cRNA 15ug (0.05ug/ul final conc.) 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.) 15ul 20x Eukaryotic Hybridization spike controls (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc. respectively) 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.) 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.) 150ul 2x Hybridization Buffer (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20) - DEPC H2O to 300ul. - Denatured at 99C 5min prior to applying to GeneChip Hybridization Conditions: - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm - 200ul denatured Hybridization cocktail is applied to GeneChip - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640. Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard Array Format. Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains filled with wash buffer A for the scanning procedure. GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol = Scanning: Automated process conducted using the Agilent GeneArray Scanner. - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip Expression protocol as follows: Pixel value = 3um Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183330/suppl/GSM183330.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183330/suppl/GSM183330.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
GSM183332 | GPL1355 |
|
KSU Dr.Lillich - IEC6 - cells treated with NSAIDS - SC1b
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells
Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings: Scaling - All Probe Sets: Target Signal = 500 Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183332
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with 1µM SC-560 for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation.
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL: Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets Prepared using the GeneChip Expression 3’-Amplificaton IVT Labeling Protocol. 30ul fragmented cRNA 15ug (0.05ug/ul final conc.) 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.) 15ul 20x Eukaryotic Hybridization spike controls (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc. respectively) 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.) 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.) 150ul 2x Hybridization Buffer (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20) - DEPC H2O to 300ul. - Denatured at 99C 5min prior to applying to GeneChip Hybridization Conditions: - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm - 200ul denatured Hybridization cocktail is applied to GeneChip - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640. Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard Array Format. Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains filled with wash buffer A for the scanning procedure. GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol = Scanning: Automated process conducted using the Agilent GeneArray Scanner. - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip Expression protocol as follows: Pixel value = 3um Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183332/suppl/GSM183332.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183332/suppl/GSM183332.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
GSM183334 | GPL1355 |
|
KSU Dr.Lillich - IEC6 - cells treated with NSAIDS - SC2b
|
IEC6 cells - ACTT Commercially available
|
Tissue: Intestinal Crypt Cells
Animal: Rat
|
Absolute and comparison analysis are conducted using the following settings: Scaling - All Probe Sets: Target Signal = 500 Normalization – User Defined: Scale Factor = 1
|
| Sample_geo_accession | GSM183334
| | Sample_status | Public on Mar 21 2009
| | Sample_submission_date | Apr 19 2007
| | Sample_last_update_date | Mar 21 2008
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Dr.Lillich, COBRE North Lab, Kansas State University
| | Sample_treatment_protocol_ch1 | Vehicle Control with 1µM SC-560 for 72 hours
| | Sample_growth_protocol_ch1 | Incubated for 72 hours at 37C in 5% CO2.
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from IEC6 cells using a RNeasy Micro kit following the manufacturer's protocol (no.74004, Qiagen; Valencia, CA).
| | Sample_label_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT)
| | Sample_label_protocol_ch1 | Affymetrix Small Sample Labeling Protocol vII (2xIVT) First Cycle of Amplification Step 1. First cycle, first strand cDNA synthesis Note: Use C thermal cycler with a heated lid for incubations in this step. Use the 4 Chold 42 minutes C 104 C 60for the addition of reagents. 70 C hold6 minutes with 1ul of4 a. Mix 1ul of total RNA (50ng/ul) C in thermal70 C holdminutes 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70 cycler with heated lid for 6 min. (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’) b. Cool to 4 c. Spin briefly. d. Add 3ul of RT-Premix-1 master mixC for 2 min. using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 0.375ul 5x first strand buffer 1ul DTT, 0.1M 0.5ul dNTP mix, 10mM 0.375ul Rnase inhibitor, 40 U/ul e.0.25ul SuperScript II, 200 U/ul 0.5ul Total Volume 3ul C 60 minutes. atIncubated at 42 f. Heat sample C for 10 minutes to inactivate SuperScript II. sample to 4 Step 2. First cycle, second strand70 g. Spin briefly and cool C. thermal cycler without heated lid forcDNA synthesis Note: Use C hold for 16 Cincubations in this step. Use 4 the addition of reagents. C 120 minutes 4 a Prepare the SS-Premix-1 as follows using the16 minutes C hold4 C 10hold components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes. 1 rxn DEPC treated water 22.75ul 5x second strand buffer 7.5ul dNTP mix, 10mM 0.75ul DNA ligase, E. coli, 10U/ul 0.25ul DNA polymerase I, E. coli 10U/ul 1ul Rnase H, 2U/ul 0.25ul Total Volume 32.5ul b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul. c. Incubate the samples at 16Mix by pipetting and spin briefly. d. C for 2 hours. e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting. f. Continue incubation at 16 for 10 minutes. Step 3. First cycle,C double-stranded cDNA cleanup by ethanol precipitation. a. Transfer the reaction to a 1.5 ml centrifuge tube. b. Add 80ul of DEPC treated water to dilute reaction. c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting. d. Precipitate e. Centrifuge at 14,000rpm forat -20 C.the double-stranded cDNA C for 2 hours. 20 minutes at 4 f. Wash pellet with 800ul of 70% cold ethanol. g. Centrifuge at minutes at 4 h. Remove ethanol and dry pellet in speed vacuum14,000rpm for 5 C. for 10 minutes. Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated. 1rxn DEPC treated water 4ul Premixed NTPs, 18.75mM each 4ul 10x reaction buffer 1ul 10x enzyme mix 1ul Total Volume 10ul b. Mix well by pipetting. Spin briefly. C for 5 hours mixing every c. Incubate at 37 Step 5. First cycle, cRNA cleanup with Qiagen30 minutes. RNeasy columns. a. Add 90ul of Rnase-free water to sample. b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook. c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution. d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm. e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis. Second Cycle of Amplification and Labeling Step 6. Second cycle, first strand cDNA synthesis. Note: Use heating blocks for incubation in this step. a. Add random primers to the cRNA sample and mix well. cRNA, 400ng 4ul Random primers, 0.2 ug/ul 1ul Total Volume 5ul C for 10 minutes b. Incubate at 70 c. Cool sample on ice for 2 minutes. d.to denature cRNA. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes. 1rxn 5x first strand buffer 2ul DTT, 0.1M 1ul dNTP mix, 10mM 0.5ul RNase Inhibitor, 40 U/ul 0.5ul SuperScript II, 200 U/ul 1ul Total Volume 5ul e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by f. Incubate the sample atpipetting. Spin briefly. C for 1 hour. Spin briefly. 42 g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the h. Heat the sample37 C for 5 minutes to inactivatesample at C for 20 minutes. at 95 RNase H. i. Cool sample on ice 2 minutes. Spin briefly. Step 7. Second cycle, second strand cDNA synthesis Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use C hold 4 C 1016 minutes C hold4 C 12070 C holdthe 4 C 6 minutesto add reagents. 4 a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the16 C holdminutes chilled sample from the previous step. b. Mix well. Spin briefly. C for 6 sample to 4 e.c. Incubate sample at 70 d. Cool the C and spin briefly.minutes. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). 1rxn DEPC treated water 43.5ul 5x second strand buffer 15ul dNTP mix, 10mM 1.5ul DNA polymerase I, E. coli, 10 U/ul 2ul Total Volume 62ul f. Add 62ul of the SS-Premix-2 to the sample making a total volume g. Incubate at 16of 75ul. Mix well by pipetting. Spin briefly. C for 2 hours. i.h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well. C for 10 Incubate at 16 minutes. Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation. a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol. b. Mix well by pipetting. c. C for 30 the samplePrecipitate the double-stranded cDNA at -20 d. Centrifuge C.minutes. at 14,000 rpm for 20 minutes at 4 e. Carefully remove supernatant and wash the Centrifuge the tube atcDNA pellet by adding 800ul of 70% cold ethanol. f. C. 14,000 rpm for 5 minutes at 4 g. Carefully remove the ethanol. Dry pellet in a atspeed vacuum for 10 minutes. h. Store dry pellet C or proceed to next step. –20 Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix). a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet. Enzo BioArray High Yield RNA Transcription Labeling kit 1rxn DEPC treated water 22ul 10X HY reaction buffer 4ul 10X Biotin labeled ribonucleotides 4ul 10X DTT 4ul 10X RNase inhibitor mix 4ul 20X T7 RNA polymerase 2ul Total Volume 40ul 1. Mix the components well after adding each reagent; especially 2.after adding water, ensuring the cDNA pellet is completely dissolved. C for Incubate at 37 5 hours mixing every 30 minutes. GeneChip Expression 3’-Amplification IVT Labeling Kit 1rxn DEPC treated water 20ul 10x IVT Labeling Buffer 4ul IVT Labeling NTP Mix 12ul IVT Labeling Enzyme Mix 4ul Total Volume 40ul 1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet is completely dissolved. 2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent condensation.
| | Sample_hyb_protocol | GENECHIP HYBRIDIZATION PROTOCOL: Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets Prepared using the GeneChip Expression 3’-Amplificaton IVT Labeling Protocol. 30ul fragmented cRNA 15ug (0.05ug/ul final conc.) 5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.) 15ul 20x Eukaryotic Hybridization spike controls (bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc. respectively) 3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.) 3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.) 150ul 2x Hybridization Buffer (Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20) - DEPC H2O to 300ul. - Denatured at 99C 5min prior to applying to GeneChip Hybridization Conditions: - GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm - 200ul denatured Hybridization cocktail is applied to GeneChip - GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640. Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard Array Format. Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C 2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains filled with wash buffer A for the scanning procedure. GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent (6xSSPE, 0.01% Tween 20) Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM MES, 0.1M [Na+], 0.01% Tween 20) 1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES, 92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O}, 24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin conjugate, 270ul DEPC H2O, 600ul total vol.) Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC 2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above}, 24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG, 3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O, 600ul total vol.) 3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.) Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains filled with wash buffer A for the scanning procedure.
| | Sample_scan_protocol = Scanning: Automated process conducted using the Agilent GeneArray Scanner. - 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip Expression protocol as follows: Pixel value = 3um Wavelength | 570nm
| | Sample_data_processing | Data was generated and calculated using GCOS1.4
| | Sample_platform_id | GPL1355
| | Sample_contact_name | Daniel,,Marcus
| | Sample_contact_email | marcus@vet.ksu.edu
| | Sample_contact_phone | 785-532-4532
| | Sample_contact_laboratory | Cellular Biophysics lab
| | Sample_contact_department | Anatomy & Physiology
| | Sample_contact_institute | Kansas State University
| | Sample_contact_address | 228 Coles Hall
| | Sample_contact_city | Manhattan
| | Sample_contact_state | KS
| | Sample_contact_zip/postal_code | 66506
| | Sample_contact_country | USA
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183334/suppl/GSM183334.CEL.gz
| | Sample_supplementary_file | ftp://ftp.ncbi.nlm.nih.gov/geo/samples/GSM183nnn/GSM183334/suppl/GSM183334.CHP.gz
| | Sample_series_id | GSE7584
| | Sample_data_row_count | 31099
| | |
|
| |
| |
Make groups for comparisons |
(2 groups will be compared at a time) |
|
| Select GSMs and click on "Add groups" |
Enter the group name here: |
|
|
|
