Tissue samples were obtained from normal female Sprague Dowley rats.
Tissue was rapidly extracted and stored in RNAlater solution (Ambion) according to the manufacturers instructions.
Total RNA was isolated and purified using RNeasy Mini Columns according to the manufacturer’s specifications. RNA quality was assessed by OD reading and was subjected to electrophoretic trace using an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA).
For target labelling, messenger RNA was amplified and labeled from 2 mg of total RNA in two steps. In the first step, mRNA was converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (IDT). In the second step, amplified and labeled cRNA (the target) was produced in an in vitro transcription reaction using T7 RNA polymerase and biotin-UTP (Affymetrix, Santa Clara, CA). Following removal of free nucleotides, target yield was measured by UV260 absorbance. For target quality assessment, approximately 200 ng of each sample cRNA target along with a control cRNA target was analyzed on the RNA 6000 LabChip using the 2100 Bioanalyzer (Agilent). The target quality was determined based on yield and cRNA size distribution produced in the in vitro synthesis reaction. Samples that fail quality control were discarded or relabeled.
For array hybridization and processing, the labelled target was fragmented at 95°C in the presence of high magnesium concentration. The fragmented material was combined with biotinylated hybridization control oligomer and biotinylated control cRNAs for BioB, BioC, BioD and cre (Affymetrix) in hybridization buffer. Ten mg of target was hybridized with the GeneChip (Rat Genome 230 2.0) array (Affymetrix) overnight, followed by washing, staining, signal amplification with biotinylated anti-streptavidin antibody, and a final staining step on the Fluidics Station (Affymetrix). The distribution of fluorescent material on the processed array was determined using the GeneArray laser scanner (Affymetrix). Image inspection was performed manually immediately following each scan.
The array image scan was processed with Affymetrix Microarray Suite software, version 5.0 (MAS 5.0). The GeneChip expression arrays contained control probe sets for both spiked and endogenous RNA transcripts (e.g., BioB, BioC, BioD, cre and species-specific actin and GAPDH).
gene expression data derived from a hypothalamus of a normal juvenile female rat. This sample is considered as a control sample.
Image processing and expression analysis were performed using Affymetrix Microarray Suite (MAS) 5.0 software. An absolute (single assay) expression analysis was performed for each GeneChip genome array hybridization. The GeneChip expression arrays contain control probe sets for both spiked and endogenous RNA transcripts (e.g., BioB, BioC, BioD, CreX and species-specific actin and GAPDH). Following image processing and absolute analysis of the array pattern with MAS 5.0, six parameters were examined to assess the overall assay performance: background, noise, average signal, % present, ratio of signal values for probe sets representing the 5’ and 3’ ends of actin and GAPDH transcripts, and total signal for BioC, BioD and CreX probe sets. Prior to further analysis, array data were globally scaled to a uniform, average target intensity for all assays prior to further analysis. Initial filters removed all absent genes and genes with no change across all data sets.