Search results for the GEO ID: GSE6197  |
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GSM ID | GPL ID |
Select for analysis |
Title |
Source name |
Description |
Characteristics |
GSM143125 | GPL341 |
|
KSU-AP-DM Rat Semi-Circular Canal Duct C1-A
|
Semi-Circular Canal Duct epithelium untreated
|
tissue: Semi-Circular Canal Duct(SCCD) Epithelia
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 | GSM143125
| | Sample_status | Public on Apr 14 2010
| | Sample_submission_date | Oct 30 2006
| | Sample_last_update_date | Apr 14 2010
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Cellular Biophysics Laboratory, Kansas State University
| | Sample_treatment_protocol_ch1 | Untreated SCCD primary culture cells
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from untreated SCCD primary culture 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 thermal cycler with a heated lid for incubations in this step. Use the 4oC hold for the addition of reagents. 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 42oC 60 minutes
| | Sample_label_protocol_ch1 | 70oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | a. Mix 1ul of total RNA (50ng/ul) with 1ul of 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70oC in thermal cycler with heated lid for 6 min.
| | Sample_label_protocol_ch1 | (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’)
| | Sample_label_protocol_ch1 | b. Cool to 4oC for 2 min.
| | 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 | e. Incubated at 42oC 60 minutes.
| | Sample_label_protocol_ch1 | f. Heat sample at 70oC for 10 minutes to inactivate SuperScript II.
| | Sample_label_protocol_ch1 | g. Spin briefly and cool sample to 4oC.
| | Sample_label_protocol_ch1 | Step 2. First cycle, second strand cDNA synthesis
| | Sample_label_protocol_ch1 | Note: Use thermal cycler without heated lid for incubations in this step. Use 4oC hold for the addition of reagents.
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate the samples at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting.
| | Sample_label_protocol_ch1 | f. Continue incubation at 16oC 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 at -20oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Centrifuge at 14,000rpm for 20 minutes at 4oC.
| | Sample_label_protocol_ch1 | f. Wash pellet with 800ul of 70% cold ethanol.
| | Sample_label_protocol_ch1 | g. Centrifuge at 14,000rpm for 5 minutes at 4oC.
| | 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 |
| | 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. Incubate at 37oC for 5 hours mixing every 30 minutes.
| | 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 | b. Incubate at 70oC for 10 minutes to denature cRNA.
| | 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 |
| | 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 | f. Incubate the sample at 42oC for 1 hour. Spin briefly.
| | Sample_label_protocol_ch1 | g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the sample at 37oC for 20 minutes.
| | Sample_label_protocol_ch1 | h. Heat the sample at 95oC for 5 minutes to inactivate 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 the 4oC hold to add reagents.
| | Sample_label_protocol_ch1 | 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate sample at 70oC for 6 minutes.
| | Sample_label_protocol_ch1 | d. Cool the sample to 4oC and spin briefly.
| | 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 | g. Incubate at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well.
| | Sample_label_protocol_ch1 | i. Incubate at 16oC for 10 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. Precipitate the double-stranded cDNA at -20oC for 30 minutes.
| | Sample_label_protocol_ch1 | d. Centrifuge the sample at 14,000 rpm for 20 minutes at 4oC.
| | 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. Centrifuge the tube at 14,000 rpm for 5 minutes at 4oC.
| | 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 at –20oC or proceed to next step.
| | 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 | 2. Incubate at 37oC for 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 Mix4ul
| | 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 | 30ulfragmented cRNA 15ug(0.05ug/ul final conc.)
| | Sample_hyb_protocol | 5ulcontrol Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.)
| | Sample_hyb_protocol | 15ul20x 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 | 3ulHerring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.)
| | Sample_hyb_protocol | 3ulAcetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.)
| | Sample_hyb_protocol | 150ul2x 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 from GCOS1.2
| | Sample_platform_id | GPL341
| | 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/GSM143nnn/GSM143125/suppl/GSM143125.CEL.gz
| | Sample_series_id | GSE6197
| | Sample_data_row_count | 15923
| | |
|
GSM143139 | GPL341 |
|
KSU-AP-DM Rat Semi-Circular Canal Duct C2-A
|
Semi-Circular Canal duct epithelium untreated
|
tissue: Semi-Circular Canal Duct (SCCD) Epithelia
animal: Rat
age:
|
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 | GSM143139
| | Sample_status | Public on Apr 14 2010
| | Sample_submission_date | Oct 30 2006
| | Sample_last_update_date | Apr 14 2010
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Cellular Biophysics Laboratory, Kansas State University
| | Sample_treatment_protocol_ch1 | Untreated SCCD primary culture cells
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from untreated SCCD primary culture 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 thermal cycler with a heated lid for incubations in this step. Use the 4oC hold for the addition of reagents. 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 42oC 60 minutes
| | Sample_label_protocol_ch1 | 70oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | a. Mix 1ul of total RNA (50ng/ul) with 1ul of 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70oC in thermal cycler with heated lid for 6 min.
| | Sample_label_protocol_ch1 | (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’)
| | Sample_label_protocol_ch1 | b. Cool to 4oC for 2 min.
| | 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 | e. Incubated at 42oC 60 minutes.
| | Sample_label_protocol_ch1 | f. Heat sample at 70oC for 10 minutes to inactivate SuperScript II.
| | Sample_label_protocol_ch1 | g. Spin briefly and cool sample to 4oC.
| | Sample_label_protocol_ch1 | Step 2. First cycle, second strand cDNA synthesis
| | Sample_label_protocol_ch1 | Note: Use thermal cycler without heated lid for incubations in this step. Use 4oC hold for the addition of reagents.
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate the samples at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting.
| | Sample_label_protocol_ch1 | f. Continue incubation at 16oC 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 at -20oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Centrifuge at 14,000rpm for 20 minutes at 4oC.
| | Sample_label_protocol_ch1 | f. Wash pellet with 800ul of 70% cold ethanol.
| | Sample_label_protocol_ch1 | g. Centrifuge at 14,000rpm for 5 minutes at 4oC.
| | 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 |
| | 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. Incubate at 37oC for 5 hours mixing every 30 minutes.
| | 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 | b. Incubate at 70oC for 10 minutes to denature cRNA.
| | 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 |
| | 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 | f. Incubate the sample at 42oC for 1 hour. Spin briefly.
| | Sample_label_protocol_ch1 | g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the sample at 37oC for 20 minutes.
| | Sample_label_protocol_ch1 | h. Heat the sample at 95oC for 5 minutes to inactivate 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 the 4oC hold to add reagents.
| | Sample_label_protocol_ch1 | 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate sample at 70oC for 6 minutes.
| | Sample_label_protocol_ch1 | d. Cool the sample to 4oC and spin briefly.
| | 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 | g. Incubate at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well.
| | Sample_label_protocol_ch1 | i. Incubate at 16oC for 10 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. Precipitate the double-stranded cDNA at -20oC for 30 minutes.
| | Sample_label_protocol_ch1 | d. Centrifuge the sample at 14,000 rpm for 20 minutes at 4oC.
| | 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. Centrifuge the tube at 14,000 rpm for 5 minutes at 4oC.
| | 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 at –20oC or proceed to next step.
| | 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 | 2. Incubate at 37oC for 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 Mix4ul
| | 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 | 30ulfragmented cRNA 15ug(0.05ug/ul final conc.)
| | Sample_hyb_protocol | 5ulcontrol Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.)
| | Sample_hyb_protocol | 15ul20x 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 | 3ulHerring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.)
| | Sample_hyb_protocol | 3ulAcetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.)
| | Sample_hyb_protocol | 150ul2x 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 from GCOS1.2
| | Sample_platform_id | GPL341
| | 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/GSM143nnn/GSM143139/suppl/GSM143139.CEL.gz
| | Sample_series_id | GSE6197
| | Sample_data_row_count | 15923
| | |
|
GSM143152 | GPL341 |
|
KSU-AP-DM Rat Semi-Circular Canal Duct C3-A
|
Semi-Circular Canal Duct epithelium untreated
|
tissue: Semi-Circular Canal Duct epithelium
animal: Rat
age:
|
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 | GSM143152
| | Sample_status | Public on Apr 14 2010
| | Sample_submission_date | Oct 30 2006
| | Sample_last_update_date | Apr 14 2010
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Cellular Biophysics Laboratory, Kansas State University
| | Sample_treatment_protocol_ch1 | Untreated SCCD primary culture cells
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from untreated SCCD primary culture 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 thermal cycler with a heated lid for incubations in this step. Use the 4oC hold for the addition of reagents. 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 42oC 60 minutes
| | Sample_label_protocol_ch1 | 70oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | a. Mix 1ul of total RNA (50ng/ul) with 1ul of 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70oC in thermal cycler with heated lid for 6 min.
| | Sample_label_protocol_ch1 | (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’)
| | Sample_label_protocol_ch1 | b. Cool to 4oC for 2 min.
| | 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 | e. Incubated at 42oC 60 minutes.
| | Sample_label_protocol_ch1 | f. Heat sample at 70oC for 10 minutes to inactivate SuperScript II.
| | Sample_label_protocol_ch1 | g. Spin briefly and cool sample to 4oC.
| | Sample_label_protocol_ch1 | Step 2. First cycle, second strand cDNA synthesis
| | Sample_label_protocol_ch1 | Note: Use thermal cycler without heated lid for incubations in this step. Use 4oC hold for the addition of reagents.
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate the samples at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting.
| | Sample_label_protocol_ch1 | f. Continue incubation at 16oC 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 at -20oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Centrifuge at 14,000rpm for 20 minutes at 4oC.
| | Sample_label_protocol_ch1 | f. Wash pellet with 800ul of 70% cold ethanol.
| | Sample_label_protocol_ch1 | g. Centrifuge at 14,000rpm for 5 minutes at 4oC.
| | 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 |
| | 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. Incubate at 37oC for 5 hours mixing every 30 minutes.
| | 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 | b. Incubate at 70oC for 10 minutes to denature cRNA.
| | 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 |
| | 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 | f. Incubate the sample at 42oC for 1 hour. Spin briefly.
| | Sample_label_protocol_ch1 | g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the sample at 37oC for 20 minutes.
| | Sample_label_protocol_ch1 | h. Heat the sample at 95oC for 5 minutes to inactivate 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 the 4oC hold to add reagents.
| | Sample_label_protocol_ch1 | 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate sample at 70oC for 6 minutes.
| | Sample_label_protocol_ch1 | d. Cool the sample to 4oC and spin briefly.
| | 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 | g. Incubate at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well.
| | Sample_label_protocol_ch1 | i. Incubate at 16oC for 10 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. Precipitate the double-stranded cDNA at -20oC for 30 minutes.
| | Sample_label_protocol_ch1 | d. Centrifuge the sample at 14,000 rpm for 20 minutes at 4oC.
| | 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. Centrifuge the tube at 14,000 rpm for 5 minutes at 4oC.
| | 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 at –20oC or proceed to next step.
| | 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 | 2. Incubate at 37oC for 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 Mix4ul
| | 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 | 30ulfragmented cRNA 15ug(0.05ug/ul final conc.)
| | Sample_hyb_protocol | 5ulcontrol Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.)
| | Sample_hyb_protocol | 15ul20x 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 | 3ulHerring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.)
| | Sample_hyb_protocol | 3ulAcetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.)
| | Sample_hyb_protocol | 150ul2x 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 from GCOS1.2
| | Sample_platform_id | GPL341
| | 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/GSM143nnn/GSM143152/suppl/GSM143152.CEL.gz
| | Sample_series_id | GSE6197
| | Sample_data_row_count | 15923
| | |
|
GSM143153 | GPL341 |
|
KSU-AP-DM Rat Semi-Circular Canal Duct C4-A
|
Semi-Circular Canal Duct epithelium untreated
|
tissue: Semi-Circular Canal Duct(SCCD) Epithelia
animal: Rat
age:
|
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 | GSM143153
| | Sample_status | Public on Apr 14 2010
| | Sample_submission_date | Oct 30 2006
| | Sample_last_update_date | Apr 14 2010
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Cellular Biophysics Laboratory, Kansas State University
| | Sample_treatment_protocol_ch1 | Untreated SCCD primary culture cells
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from untreated SCCD primary culture 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 thermal cycler with a heated lid for incubations in this step. Use the 4oC hold for the addition of reagents. 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 42oC 60 minutes
| | Sample_label_protocol_ch1 | 70oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | a. Mix 1ul of total RNA (50ng/ul) with 1ul of 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70oC in thermal cycler with heated lid for 6 min.
| | Sample_label_protocol_ch1 | (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’)
| | Sample_label_protocol_ch1 | b. Cool to 4oC for 2 min.
| | 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 | e. Incubated at 42oC 60 minutes.
| | Sample_label_protocol_ch1 | f. Heat sample at 70oC for 10 minutes to inactivate SuperScript II.
| | Sample_label_protocol_ch1 | g. Spin briefly and cool sample to 4oC.
| | Sample_label_protocol_ch1 | Step 2. First cycle, second strand cDNA synthesis
| | Sample_label_protocol_ch1 | Note: Use thermal cycler without heated lid for incubations in this step. Use 4oC hold for the addition of reagents.
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate the samples at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting.
| | Sample_label_protocol_ch1 | f. Continue incubation at 16oC 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 at -20oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Centrifuge at 14,000rpm for 20 minutes at 4oC.
| | Sample_label_protocol_ch1 | f. Wash pellet with 800ul of 70% cold ethanol.
| | Sample_label_protocol_ch1 | g. Centrifuge at 14,000rpm for 5 minutes at 4oC.
| | 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 |
| | 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. Incubate at 37oC for 5 hours mixing every 30 minutes.
| | 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 | b. Incubate at 70oC for 10 minutes to denature cRNA.
| | 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 |
| | 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 | f. Incubate the sample at 42oC for 1 hour. Spin briefly.
| | Sample_label_protocol_ch1 | g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the sample at 37oC for 20 minutes.
| | Sample_label_protocol_ch1 | h. Heat the sample at 95oC for 5 minutes to inactivate 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 the 4oC hold to add reagents.
| | Sample_label_protocol_ch1 | 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate sample at 70oC for 6 minutes.
| | Sample_label_protocol_ch1 | d. Cool the sample to 4oC and spin briefly.
| | 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 | g. Incubate at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well.
| | Sample_label_protocol_ch1 | i. Incubate at 16oC for 10 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. Precipitate the double-stranded cDNA at -20oC for 30 minutes.
| | Sample_label_protocol_ch1 | d. Centrifuge the sample at 14,000 rpm for 20 minutes at 4oC.
| | 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. Centrifuge the tube at 14,000 rpm for 5 minutes at 4oC.
| | 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 at –20oC or proceed to next step.
| | 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 | 2. Incubate at 37oC for 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 Mix4ul
| | 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 | 30ulfragmented cRNA 15ug(0.05ug/ul final conc.)
| | Sample_hyb_protocol | 5ulcontrol Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.)
| | Sample_hyb_protocol | 15ul20x 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 | 3ulHerring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.)
| | Sample_hyb_protocol | 3ulAcetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.)
| | Sample_hyb_protocol | 150ul2x 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 from GCOS1.2
| | Sample_platform_id | GPL341
| | 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/GSM143nnn/GSM143153/suppl/GSM143153.CEL.gz
| | Sample_series_id | GSE6197
| | Sample_data_row_count | 15923
| | |
|
GSM143154 | GPL341 |
|
KSU-AP-DM Rat Semi-Circular Canal Duct D1-A
|
Semi-Circular Canal Duct epithelium - Dex treated
|
tissue: Semi-Circular Canal Duct epithelium - Dex treated
animal: Rat
age:
|
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 | GSM143154
| | Sample_status | Public on Apr 14 2010
| | Sample_submission_date | Oct 30 2006
| | Sample_last_update_date | Apr 14 2010
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Cellular Biophysics Laboratory, Kansas State University
| | Sample_treatment_protocol_ch1 | SCCD primary culture cells treated with 100nM Dexamethasone
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from SCCD primary culture cells treated with 100 nM Dexamethasone 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 thermal cycler with a heated lid for incubations in this step. Use the 4oC hold for the addition of reagents. 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 42oC 60 minutes
| | Sample_label_protocol_ch1 | 70oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | a. Mix 1ul of total RNA (50ng/ul) with 1ul of 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70oC in thermal cycler with heated lid for 6 min.
| | Sample_label_protocol_ch1 | (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’)
| | Sample_label_protocol_ch1 | b. Cool to 4oC for 2 min.
| | 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 | e. Incubated at 42oC 60 minutes.
| | Sample_label_protocol_ch1 | f. Heat sample at 70oC for 10 minutes to inactivate SuperScript II.
| | Sample_label_protocol_ch1 | g. Spin briefly and cool sample to 4oC.
| | Sample_label_protocol_ch1 | Step 2. First cycle, second strand cDNA synthesis
| | Sample_label_protocol_ch1 | Note: Use thermal cycler without heated lid for incubations in this step. Use 4oC hold for the addition of reagents.
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate the samples at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting.
| | Sample_label_protocol_ch1 | f. Continue incubation at 16oC 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 at -20oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Centrifuge at 14,000rpm for 20 minutes at 4oC.
| | Sample_label_protocol_ch1 | f. Wash pellet with 800ul of 70% cold ethanol.
| | Sample_label_protocol_ch1 | g. Centrifuge at 14,000rpm for 5 minutes at 4oC.
| | 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 |
| | 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. Incubate at 37oC for 5 hours mixing every 30 minutes.
| | 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 | b. Incubate at 70oC for 10 minutes to denature cRNA.
| | 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 |
| | 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 | f. Incubate the sample at 42oC for 1 hour. Spin briefly.
| | Sample_label_protocol_ch1 | g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the sample at 37oC for 20 minutes.
| | Sample_label_protocol_ch1 | h. Heat the sample at 95oC for 5 minutes to inactivate 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 the 4oC hold to add reagents.
| | Sample_label_protocol_ch1 | 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate sample at 70oC for 6 minutes.
| | Sample_label_protocol_ch1 | d. Cool the sample to 4oC and spin briefly.
| | 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 | g. Incubate at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well.
| | Sample_label_protocol_ch1 | i. Incubate at 16oC for 10 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. Precipitate the double-stranded cDNA at -20oC for 30 minutes.
| | Sample_label_protocol_ch1 | d. Centrifuge the sample at 14,000 rpm for 20 minutes at 4oC.
| | 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. Centrifuge the tube at 14,000 rpm for 5 minutes at 4oC.
| | 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 at –20oC or proceed to next step.
| | 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 | 2. Incubate at 37oC for 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 Mix4ul
| | 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 | 30ulfragmented cRNA 15ug(0.05ug/ul final conc.)
| | Sample_hyb_protocol | 5ulcontrol Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.)
| | Sample_hyb_protocol | 15ul20x 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 | 3ulHerring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.)
| | Sample_hyb_protocol | 3ulAcetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.)
| | Sample_hyb_protocol | 150ul2x 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 from GCOS1.2
| | Sample_platform_id | GPL341
| | 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/GSM143nnn/GSM143154/suppl/GSM143154.CEL.gz
| | Sample_series_id | GSE6197
| | Sample_data_row_count | 15923
| | |
|
GSM143155 | GPL341 |
|
KSU-AP-DM Rat Semi-Circular Canal Duct D2-A
|
Semi-Circular Canal Duct Epithelium - Dex treated
|
tissue: Semi-Circular Canal Duct Epithelium - Dex treated
animal: Rat
age:
|
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 | GSM143155
| | Sample_status | Public on Apr 14 2010
| | Sample_submission_date | Oct 30 2006
| | Sample_last_update_date | Apr 14 2010
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Cellular Biophysics Laboratory, Kansas State University
| | Sample_treatment_protocol_ch1 | SCCD primary culture cells treated with 100nM Dexamethasone
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from SCCD primary culture cells treated with 100 nM Dexamethasone 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 thermal cycler with a heated lid for incubations in this step. Use the 4oC hold for the addition of reagents. 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 42oC 60 minutes
| | Sample_label_protocol_ch1 | 70oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | a. Mix 1ul of total RNA (50ng/ul) with 1ul of 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70oC in thermal cycler with heated lid for 6 min.
| | Sample_label_protocol_ch1 | (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’)
| | Sample_label_protocol_ch1 | b. Cool to 4oC for 2 min.
| | 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 | e. Incubated at 42oC 60 minutes.
| | Sample_label_protocol_ch1 | f. Heat sample at 70oC for 10 minutes to inactivate SuperScript II.
| | Sample_label_protocol_ch1 | g. Spin briefly and cool sample to 4oC.
| | Sample_label_protocol_ch1 | Step 2. First cycle, second strand cDNA synthesis
| | Sample_label_protocol_ch1 | Note: Use thermal cycler without heated lid for incubations in this step. Use 4oC hold for the addition of reagents.
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate the samples at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting.
| | Sample_label_protocol_ch1 | f. Continue incubation at 16oC 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 at -20oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Centrifuge at 14,000rpm for 20 minutes at 4oC.
| | Sample_label_protocol_ch1 | f. Wash pellet with 800ul of 70% cold ethanol.
| | Sample_label_protocol_ch1 | g. Centrifuge at 14,000rpm for 5 minutes at 4oC.
| | 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 |
| | 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. Incubate at 37oC for 5 hours mixing every 30 minutes.
| | 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 | b. Incubate at 70oC for 10 minutes to denature cRNA.
| | 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 |
| | 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 | f. Incubate the sample at 42oC for 1 hour. Spin briefly.
| | Sample_label_protocol_ch1 | g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the sample at 37oC for 20 minutes.
| | Sample_label_protocol_ch1 | h. Heat the sample at 95oC for 5 minutes to inactivate 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 the 4oC hold to add reagents.
| | Sample_label_protocol_ch1 | 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate sample at 70oC for 6 minutes.
| | Sample_label_protocol_ch1 | d. Cool the sample to 4oC and spin briefly.
| | 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 | g. Incubate at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well.
| | Sample_label_protocol_ch1 | i. Incubate at 16oC for 10 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. Precipitate the double-stranded cDNA at -20oC for 30 minutes.
| | Sample_label_protocol_ch1 | d. Centrifuge the sample at 14,000 rpm for 20 minutes at 4oC.
| | 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. Centrifuge the tube at 14,000 rpm for 5 minutes at 4oC.
| | 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 at –20oC or proceed to next step.
| | 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 | 2. Incubate at 37oC for 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 Mix4ul
| | 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 | 30ulfragmented cRNA 15ug(0.05ug/ul final conc.)
| | Sample_hyb_protocol | 5ulcontrol Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.)
| | Sample_hyb_protocol | 15ul20x 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 | 3ulHerring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.)
| | Sample_hyb_protocol | 3ulAcetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.)
| | Sample_hyb_protocol | 150ul2x 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 from GCOS1.2
| | Sample_platform_id | GPL341
| | 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/GSM143nnn/GSM143155/suppl/GSM143155.CEL.gz
| | Sample_series_id | GSE6197
| | Sample_data_row_count | 15923
| | |
|
GSM143157 | GPL341 |
|
KSU-AP-DM Rat Semi-Circular Canal Duct D3-A
|
Semi-Circular Canal Duct epithelium - Dex treated
|
tissue: Semi-Circular Canal Duct (SCCD) Epithelia
animal: Rat
age:
|
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 | GSM143157
| | Sample_status | Public on Apr 14 2010
| | Sample_submission_date | Oct 30 2006
| | Sample_last_update_date | Apr 14 2010
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Cellular Biophysics Laboratory, Kansas State University
| | Sample_treatment_protocol_ch1 | SCCD primary culture cells treated with 100nM Dexamethasone
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from SCCD primary culture cells treated with 100 nM Dexamethasone 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 thermal cycler with a heated lid for incubations in this step. Use the 4oC hold for the addition of reagents. 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 42oC 60 minutes
| | Sample_label_protocol_ch1 | 70oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | a. Mix 1ul of total RNA (50ng/ul) with 1ul of 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70oC in thermal cycler with heated lid for 6 min.
| | Sample_label_protocol_ch1 | (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’)
| | Sample_label_protocol_ch1 | b. Cool to 4oC for 2 min.
| | 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 | e. Incubated at 42oC 60 minutes.
| | Sample_label_protocol_ch1 | f. Heat sample at 70oC for 10 minutes to inactivate SuperScript II.
| | Sample_label_protocol_ch1 | g. Spin briefly and cool sample to 4oC.
| | Sample_label_protocol_ch1 | Step 2. First cycle, second strand cDNA synthesis
| | Sample_label_protocol_ch1 | Note: Use thermal cycler without heated lid for incubations in this step. Use 4oC hold for the addition of reagents.
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate the samples at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting.
| | Sample_label_protocol_ch1 | f. Continue incubation at 16oC 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 at -20oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Centrifuge at 14,000rpm for 20 minutes at 4oC.
| | Sample_label_protocol_ch1 | f. Wash pellet with 800ul of 70% cold ethanol.
| | Sample_label_protocol_ch1 | g. Centrifuge at 14,000rpm for 5 minutes at 4oC.
| | 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 |
| | 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. Incubate at 37oC for 5 hours mixing every 30 minutes.
| | 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 | b. Incubate at 70oC for 10 minutes to denature cRNA.
| | 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 |
| | 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 | f. Incubate the sample at 42oC for 1 hour. Spin briefly.
| | Sample_label_protocol_ch1 | g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the sample at 37oC for 20 minutes.
| | Sample_label_protocol_ch1 | h. Heat the sample at 95oC for 5 minutes to inactivate 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 the 4oC hold to add reagents.
| | Sample_label_protocol_ch1 | 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate sample at 70oC for 6 minutes.
| | Sample_label_protocol_ch1 | d. Cool the sample to 4oC and spin briefly.
| | 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 | g. Incubate at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well.
| | Sample_label_protocol_ch1 | i. Incubate at 16oC for 10 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. Precipitate the double-stranded cDNA at -20oC for 30 minutes.
| | Sample_label_protocol_ch1 | d. Centrifuge the sample at 14,000 rpm for 20 minutes at 4oC.
| | 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. Centrifuge the tube at 14,000 rpm for 5 minutes at 4oC.
| | 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 at –20oC or proceed to next step.
| | 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 | 2. Incubate at 37oC for 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 Mix4ul
| | 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 | 30ulfragmented cRNA 15ug(0.05ug/ul final conc.)
| | Sample_hyb_protocol | 5ulcontrol Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.)
| | Sample_hyb_protocol | 15ul20x 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 | 3ulHerring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.)
| | Sample_hyb_protocol | 3ulAcetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.)
| | Sample_hyb_protocol | 150ul2x 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 from GCOS1.2
| | Sample_platform_id | GPL341
| | 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/GSM143nnn/GSM143157/suppl/GSM143157.CEL.gz
| | Sample_series_id | GSE6197
| | Sample_data_row_count | 15923
| | |
|
GSM143162 | GPL341 |
|
KSU-AP-DM Rat Semi-Circular Canal Duct D4-A
|
Semi-Circular Canal Duct epithelium - Dex treated
|
tissue: Semi-Circular Canal Duct (SCCD) Epithelia
animal: Rat
age:
|
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 | GSM143162
| | Sample_status | Public on Apr 14 2010
| | Sample_submission_date | Oct 30 2006
| | Sample_last_update_date | Apr 14 2010
| | Sample_type | RNA
| | Sample_channel_count | 1
| | Sample_organism_ch1 | Rattus norvegicus
| | Sample_taxid_ch1 | 10116
| | Sample_biomaterial_provider_ch1 | Cellular Biophysics Laboratory, Kansas State University
| | Sample_treatment_protocol_ch1 | SCCD primary culture cells treated with 100nM Dexamethasone
| | Sample_molecule_ch1 | total RNA
| | Sample_extract_protocol_ch1 | Total RNA was extracted from SCCD primary culture 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 thermal cycler with a heated lid for incubations in this step. Use the 4oC hold for the addition of reagents. 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 42oC 60 minutes
| | Sample_label_protocol_ch1 | 70oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | a. Mix 1ul of total RNA (50ng/ul) with 1ul of 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70oC in thermal cycler with heated lid for 6 min.
| | Sample_label_protocol_ch1 | (5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’)
| | Sample_label_protocol_ch1 | b. Cool to 4oC for 2 min.
| | 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 | e. Incubated at 42oC 60 minutes.
| | Sample_label_protocol_ch1 | f. Heat sample at 70oC for 10 minutes to inactivate SuperScript II.
| | Sample_label_protocol_ch1 | g. Spin briefly and cool sample to 4oC.
| | Sample_label_protocol_ch1 | Step 2. First cycle, second strand cDNA synthesis
| | Sample_label_protocol_ch1 | Note: Use thermal cycler without heated lid for incubations in this step. Use 4oC hold for the addition of reagents.
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate the samples at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting.
| | Sample_label_protocol_ch1 | f. Continue incubation at 16oC 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 at -20oC for 2 hours.
| | Sample_label_protocol_ch1 | e. Centrifuge at 14,000rpm for 20 minutes at 4oC.
| | Sample_label_protocol_ch1 | f. Wash pellet with 800ul of 70% cold ethanol.
| | Sample_label_protocol_ch1 | g. Centrifuge at 14,000rpm for 5 minutes at 4oC.
| | 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 |
| | 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. Incubate at 37oC for 5 hours mixing every 30 minutes.
| | 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 | b. Incubate at 70oC for 10 minutes to denature cRNA.
| | 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 |
| | 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 | f. Incubate the sample at 42oC for 1 hour. Spin briefly.
| | Sample_label_protocol_ch1 | g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the sample at 37oC for 20 minutes.
| | Sample_label_protocol_ch1 | h. Heat the sample at 95oC for 5 minutes to inactivate 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 the 4oC hold to add reagents.
| | Sample_label_protocol_ch1 | 70oC 6 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 120 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | Sample_label_protocol_ch1 | 16oC 10 minutes
| | Sample_label_protocol_ch1 | 4oC hold
| | 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. Incubate sample at 70oC for 6 minutes.
| | Sample_label_protocol_ch1 | d. Cool the sample to 4oC and spin briefly.
| | 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 | g. Incubate at 16oC for 2 hours.
| | Sample_label_protocol_ch1 | h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well.
| | Sample_label_protocol_ch1 | i. Incubate at 16oC for 10 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. Precipitate the double-stranded cDNA at -20oC for 30 minutes.
| | Sample_label_protocol_ch1 | d. Centrifuge the sample at 14,000 rpm for 20 minutes at 4oC.
| | 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. Centrifuge the tube at 14,000 rpm for 5 minutes at 4oC.
| | 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 at –20oC or proceed to next step.
| | 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 | 2. Incubate at 37oC for 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 Mix4ul
| | 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 | 30ulfragmented cRNA 15ug(0.05ug/ul final conc.)
| | Sample_hyb_protocol | 5ulcontrol Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.)
| | Sample_hyb_protocol | 15ul20x 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 | 3ulHerring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.)
| | Sample_hyb_protocol | 3ulAcetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.)
| | Sample_hyb_protocol | 150ul2x 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 GCOS1.2
| | Sample_platform_id | GPL341
| | 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/GSM143nnn/GSM143162/suppl/GSM143162.CEL.gz
| | Sample_series_id | GSE6197
| | Sample_data_row_count | 15923
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