|
| Status |
Public on Mar 04, 2008 |
| Title |
T0004 (parental) vs 43264 (congenic) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
T0004 (Inbred Dahl salt-sensitive (SS/Jr) rat )
|
| Organism |
Rattus norvegicus |
| Characteristics |
Inbred Dahl salt-sensitive (SS/Jr) adult male rat; tissue: kidney
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue was processed using Trizol reagent (Invitrogen), and total RNA was extracted using RNeasy columns (Qiagen) according to manufacturer's protocols.
|
| Label |
Cy3
|
| Label protocol |
Single-stranded cDNA was generated through reverse transcription of total RNA by Superscript III (Invitrogen) and random primers. Amino-allyl dUTP (in a ratio of 2:3 with dTTP) was incorporated during the reverse transcription. The RNA was then degraded by hydrolysis under alkaline conditions and the amino-allyl cDNA was subsequently purified with PCR Purification columns (Qiagen) substituting a phosphate wash buffer and phosphate elution buffer for the Tris-containing buffers in the kit. The purified sample was lyophilized and resuspended in sodium carbonate buffer and allowed to couple to the Cy3 fluorophore for an hour at room temperature. The reaction was stopped by sodium acetate and then the labeled cDNA was purified to remove uncoupled dye with PCR Purification columns (Qiagen) as per manufacturer's protocol.
|
| |
|
| Channel 2 |
| Source name |
43264 ( S.LEW (5)x6x9 )
|
| Organism |
Rattus norvegicus |
| Characteristics |
Congenic strain S.LEW (5)x6x9 adult male rat; tissue: kidney
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue was processed using Trizol reagent (Invitrogen), and total RNA was extracted using RNeasy columns (Qiagen) according to manufacturer's protocols.
|
| Label |
Cy5
|
| Label protocol |
Single-stranded cDNA was generated through reverse transcription of total RNA by Superscript III (Invitrogen) and random primers. Amino-allyl dUTP (in a ratio of 2:3 with dTTP) was incorporated during the reverse transcription. The RNA was then degraded by hydrolysis under alkaline conditions and the amino-allyl cDNA was subsequently purified with PCR Purification columns (Qiagen) substituting a phosphate wash buffer and phosphate elution buffer for the Tris-containing buffers in the kit. The purified sample was lyophilized and resuspended in sodium carbonate buffer and allowed to couple to the Cy5 fluorophore for an hour at room temperature. The reaction was stopped by sodium acetate and then the labeled cDNA was purified to remove uncoupled dye with PCR Purification columns (Qiagen) as per manufacturer's protocol.
|
| |
|
| |
| Hybridization protocol |
Printed arrays were pre-hybridized in a prehybridization buffer (5x SSC; 0.1% SDS; 1% BSA) for 45 minutes at 42C. Labeled cDNA samples (one labeled with Cy3 and a second with Cy5) were combined and lyophilized. The lyophilized pellet was resuspended in a hybridization buffer (50% formamide; 5x SSC; 0.1% SDS) and heated to 95C for 5 minutes and immediately snap-cooled. The labeled cDNA was then applied to the pre-hybridzed array under glass coverslip. The array was sealed in a hyb chamber and incubated overnight at 42C. The array was subsequently washed three times in buffers of increasing stringency (Low: 1.0x SSC, 0.2% SDS; Medium: 0.1x SSC, 0.2% SDS; High: 0.1x SSC). The arrays were quickly dried and immediately scanned.
|
| Scan protocol |
The hybridized microarray chips were scanned on an Axon GenePix 4000B scanner under the two-color mode for Cy3 and Cy5 fluorophores.
|
| Description |
T0004 (parental) vs 43264 (congenic)
|
| Data processing |
The Cy3 and Cy5 images were analyzed through GenePix Pro 4.0 to calculate spot intensities and exported obtaining a .gpr file. The .gpr file was run through TIGR Express Converter to generate a .mev file. The .mev file was through the TIGR MIDAS using a lowess normalization.
|
| |
|
| Submission date |
Apr 26, 2007 |
| Last update date |
Mar 03, 2008 |
| Contact name |
Norman H Lee |
| E-mail(s) |
phmnhl@gwumc.edu
|
| Organization name |
The George Washington University Medical Center
|
| Department |
Pharmacology & Physiology
|
| Street address |
2300 I Street, N.W.
|
| City |
Washington, DC |
| State/province |
DC |
| ZIP/Postal code |
20037 |
| Country |
USA |
| |
|
| Platform ID |
GPL5126 |
| Series (1) |
| GSE7628 |
Evaluation of expression quantitative trait loci within two interacting blood pressure quantitative trait loci |
|
