Ovation exon module kit

After validation of RNA quality with the Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit), 50 ng of total RNA were reverse transcribed following the Ovation PicoSL WTA System (Nugen). Briefly, the resulting double-strand cDNA was used for amplification based on SPIA technology. After purification according to the Nugen protocol, 5 μg of single strand DNA were used for generation of Sens Target DNA using the Ovation Exon Module kit (Nugen). 2.5 μg of Sens Target DNA were fragmented and labelled with biotin using Encore Biotin Module kit (Nugen). After control of fragmentation using a Bioanalyzer 2100, the cDNA was then hybridized to GeneChip^® Mouse Gene 1.0 ST (Affymetrix) at 45°C for 17 h. After overnight hybridization, the ChIPs were washed using the fluidic station FS450 following specific protocols (Affymetrix) and scanned using the GCS3000 7G. The scanned images were then analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files) and metrics for Quality Controls. The analysis of some of these metrics and the study of the distribution of raw data show no out-lier experiments. RMA normalization was performed using R and normalized data were subjected to statistical tests.