Abstract
RNA integrity is one of the most important factors influencing the generation of accurate quantitative results from gene expression experiments. Degraded RNA, such as that typically derived from formalin-fixed paraffin embedded (FFPE) tissue, can hinder amplification and labeling of cDNA. In addition to the direct hybridization whole-genome expression method, Illumina offers the whole-genome cDNA-mediated annealing, selection, extension, and ligation (DASL) assay for expression profiling of partially degraded RNA. Here we purposely degrade RNA to demonstrate the effects of RNA degradation on the performance of the DASL vs. direct hybridization method. MCF7 cell lines were exposed to either 5uM or 10uM ERRbeta/ERRgamma agonist DY131, or DMSO control. RNA was isolated, and fully intact RNA (495 ng, RIN = 10) was processed using the direct hybridization method and hybridized to a HumanHT-12 Expression BeadChip in duplicate. Treatment samples (5uM or 10uM DY131) were compared to the control (DMSO) sample and analyzed for differentially expressed genes (DEGs). The same RNAs (495 ng, RIN = 10) were processed in parallel using the DASL method and analyzed as above. The same RNA samples were then incubated for 5 minutes at 90°C to degrade to an average RIN = 6.1 and processed with the DASL method and analyzed as above. Using the direct hybridization and DASL methods with fully intact RNA, a total of 138 and 165 genes, respectively, were found to be differentially expressed after 5uM or 10uM DY131 incubation vs. control (DMSO) (p-value < 0.05, 2-fold change cutoff, one-way ANOVA). However, only 34 of these genes overlapped. Analysis of degraded RNA (DASL) revealed 165 DEGs, with 98 overlapping with DEGs found in fully intact RNA (DASL). Overall, we found that although the DASL method increases sensitivity, it is challenged to maintain similar expression patterns with the direct hybridization method for both fully intact and partially degraded RNA.