strain: S.R(ET3x2), diet: high salt (Harlan Teklad TD83033, 4% NaCl), age: 40-42 days, gender: male, tissue: left kidney
Treatment protocol
Age-matched male S (n=10), S.R(ET3x1) (n=8), and S.R(ET3x2) (n=8) rats were bred, housed, and studied concomitantly to minimize envi¬ronmental effects. Rats were weaned at 30 days of age onto a low salt diet (0.4% NaCl, Harlan Teklad diet TD7034) and water ad libitum. At 39-41 days of age one-half of rats from each strain group were switched to a 4% NaCl diet (Harlan Teklad diet TD83033) and water ad libitum for 24 hours. All rats were then killed by pentobarbital overdose. Kidneys were removed, decapsulated, blotted dry, and weighed. The kidneys of each rat were then processed separately for isolation of total RNA.
Extracted molecule
total RNA
Extraction protocol
A single rat kidney RNA sample was used for each of 26 RAE230A/B chipsets [10 S-rat samples, 8 S.R(ET3x1) samples, and 8 S.R(ET3x2) samples]. Total RNA was isolated from the kidneys of each rat separately, essentially as described (Lee et al 2003). Briefly, kidneys were homogenized in a guanidine thiocyanate/phenol solution (Ultraspec, Biotecx Laboratories, Houston, TX), extracted with chloroform and isolated using RNA Tack Resin (Biotecx Laboratories). Total RNA was further purified by ethanol precipitation and absorption to a column (RNeasy, Qiagen, Valencia, CA) following the manufacturer's instructions. Kidney RNA integrity was assessed by 1) electrophoretic size-fractionation on 1% agarose gels under denaturing conditions and 2) hybridization of newly made cRNA probes to TestChips (Affymetrix).
Label
biotin
Label protocol
Oligonucleotide microarray analysis was performed essentially as described in Lee et al. (2005) using the Affymetrix Rat Genome RAE230 array set (GeneChips 230A and 230B) according to the manufacturer’s protocol. Double-stranded cDNA was synthesized from 15 microgram total RNA from each rat kidney RNA preparation using reverse transcriptase (SuperScript II, InVitrogen, Carlsbad, CA) and T7-(dT)24 as the oligonucleotide primer. Biotinylated cRNA was synthesized using a kit (Enzo Bioarray High Yield RNA transcript Labeling Kit, Enzo Diagnostics, Farmingdale, NY) and cRNAs were purified on columns (RNeasy mini kit, Qiagen).
Hybridization protocol
Each cRNA was fragmented according to the protocol in the Affymetrix GeneChip Expression Analysis manual with quality assessed by hybridization of a 5 g aliquot to a test chip (TestChip3, Affymetrix). Fragmented cRNA probe (5 g) was then hybridized to each of a set of 2 rat GeneChips (RAE230A and RAE230B; Affymetrix).
Scan protocol
Scanning was performed at the University of Toledo Health Science Campus Genomics Core Facility according to the manufacturer's (Affymetrix) instructions.
Description
Gene expression data from S.R(ET3x2) rat 39801 left kidney after 24 hours on high salt diet
Data processing
Probe set expression measures were calculated from probe-level data using RMAExpress v0.5 software with background adjustment, quantile normalization and log 2 transformation
