The genetic expression profile for one male and one female continuous GH treated male liver sample is given as a lowess normalized ratio (f=0.2). This result is from a single random pairing of one of four males and one of four GH treated males. The purpose of this pairwise comparison is to highlight the GH-dependent differences in rat liver gene expression.
Technical Aspects: Adult male and female Fischer 344 rats (8-10 weeks of age) were untreated or were treated with rat growth hormone (GH) (NIDDK, rGH-B-14-SIAFP) given as a continuous infusion using an Alzet osmotic minipump delivering 2 ug GH/100g body weight/h for 7 days. Intact male and female rats were killed and their livers were collected, frozen in liquid N2 and stored at –80ºC. Total liver RNA was isolated from frozen liver samples using Trizol reagent (Invitrogen). Poly(A) RNA was then isolated using a commercial kit (Ambion). RNA samples having an A260/A280 ratio >= 1.8 were deemed suitable for use in subsequent analyses. Total liver RNA and poly(A) RNA samples were treated with DNase I (Ambion). Poly(A) RNA purified from individual livers was labeled with Cy3-dUTP or Cy5-dUTP (PerkinElmer) in a reverse transcription reaction using 1 ug poly(A) RNA and Superscript II (RNase H mutant) enzyme (Invitrogen) at 39oC for 2 hr. Unincorporated dNTPs were removed using the Qiaquick PCR-purification kit (Qiagen). Cy5-labeled male liver cDNA was mixed with Cy3-labeled female or Cy3-labeled GH-treated male liver cDNA followed by co-hybridization of the mixed cDNA population to the microarray for 18 hr at 42oC. Arrays were washed according to the manufacturer’s protocol. RNA from each of four male, female, and GH-treated male livers was converted to cDNA and labeled in duplicate. One series of eight hybridizations (four male + female liver cDNA pairings, and four male + male with GH cDNA pairings) was carried out on MWG Rat Liver arrays. A second series containing the same eight hybridizations was carried out on MWG Rat 5K arrays. The fluorescent cDNA on each microarray was detected using a GenePix 4000B array scanner (Axon Instruments). The data was preprocessed using GenePix Pro 3.0 (Axon Instruments) and analyzed in GeneSpring 6.0 (Silicon Genetics). Any spot where there was no fluorescence is reported as 0. The normalized average ratio is given for all duplicates.
