Total RNA from cells was isolated using an RNeasy system (Qiagen, Valencia, CA, USA) and were further purified by passage through RNeasy mini-columns (QIAGEN, Valencia, CA) according to manufacturer’s protocols for RNA clean-up. Final RNA preparations were resuspended in RNase-free water and stored at -80°C. The yield of the extracted RNA was determined spectrophotometrically by measuring the optical density at 260 nm. The purity and quality of extracted RNA were evaluated using the RNA 6000 LabChip and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Only high-quality RNA with RNA integrity numbers (RINs) greater than 7.5 were used for microarray experiments
Label
Digoxigenin-labelled cRNA
Label protocol
All RNA targets were labelled using the Applied Biosystems RT-IVT Labelling Kit Version 2.0. Briefly, 1.5 μg of the total RNA sample was reverse transcribed via 2 h incubation at 42 ◦C with ArrayScript RT enzyme (Ambion, Austin, TX, USA) and oligo dT-T7 primer. Double-stranded cDNA was produced following 2 h incubation with E. coli DNA polymerase and RNase H at 16 ◦C. Double-stranded cDNA was purified according to the RT-IVT kit protocol. In vitro transcription was performed by incubation of the cDNA product with T7 RNA polymerase, 0.75 mM digoxigenin-11-UTP (Roche Applied Science, Indianapolis, IN, USA) and all other NTPs for 9 h. Labelled cRNA was purified according to the RT-IVT kit protocol and analyzed for quality and quantity using standard UV spectrometry and the Bioanalyzer.
Hybridization protocol
Digoxigenin-labelled cRNA targets were hybridized to Applied Biosystems Rat Whole Genome Survey Microarrays using the Applied Biosystems Chemiluminescent Detection Kit. Briefly, 15 μg of labelled cRNA targets were fragmented via incubation for 30 min at 60 ◦C with fragmentation buffer provided in the kit. Fragmented targets were hybridized to microarrays during 16 h of incubation at 55 ◦C with buffers and reagents from the Chemiluminescent Detection Kit. Post-hybridization washes and anti-digoxigenin-alkaline phosphatase binding were performed according to the kit protoco
Scan protocol
Chemiluminescence detection, image acquisition and analysis were performed using the Applied Biosystems Chemiluminescence Detection Kit and Applied Biosystems 1700 Chemiluminescent Microarray Analyzer following the manufacturer’s protocols. Images were autogridded and the chemiluminescent signals were quantified, corrected for background and, finally, spot- and spatially normalized using the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer software version 1.1 (for more information see Applied Biosystems 1700 Chemiluminescent Microarray Analyzer user bulletin in ABI's website)
Description
Tissue from Brown Norway rat strain
Data processing
The data obtained after the process described in the scan protocol section were normalized by LOWESS (Locally weighted least mean squares scatter Plot smoother), this was the statistical algorithm for performing a non linear normalization and corrects deviations of the backbone of the data cloud from the expected main diagonal. Data were LOWESS normalized excluding the controls on the microarray and using only the transcripts with a S/N ratio to > 2.
