Gene expression profiling of samples from biobanks requires a method that can be used with intact as well as partially degraded RNA. High throughput applications can benefit from reducing the number of processing steps including eliminating the poly(A) selection and ribosomal depletion steps. When performing targeted capture, we have found that we can eliminate the upfront poly(A) selection/ribosomal depletion steps that cause bias in standard mRNA-Seq workflows. This target enrichment solution allows for whole transcriptome or customized content to characterize differential gene expression patterns (especially for mid/low level transcripts). Protocol modifications to the Agilent Strand-Specific RNA Library Prep kit resulted in a new workflow called "RNA Direct" that generates RNA-Seq data with minimal ribosomal contamination and good sequencing coverage. Using RNA isolated from a set of matched samples including fresh frozen (FF) or formalin-fixed, paraffin-embedded (FFPE) from tumor/normal tissues we generated high-quality data using a protocol that does not require upfront ribosomal depletion or poly(A) selection. Using SureSelectXT RNA Direct protocol (RNA Direct) workflow, we found transcripts to be upregulated or downregulated to similar degrees with similar confidence levels in both the FF and FFPE samples, demonstrating the utility for meaningful gene expression studies with biobank samples of variable quality.
Keywords: Capture probes; Gene expression; Next generation sequencing; RNA-Seq; Target enrichment; Transcriptome; Tumor profiling.