RNA sequencing (RNA-Seq) is often used for transcriptome profiling as well as the identification of novel transcripts and alternative splicing events. Typically, RNA-Seq libraries are prepared from total RNA using poly(A) enrichment of the mRNA (mRNA-Seq) to remove ribosomal RNA (rRNA), however, this method fails to capture non-poly(A) transcripts or partially degraded mRNAs. Hence, a mRNA-Seq protocol will not be compatible for use with RNAs coming from Formalin-Fixed and Paraffin-Embedded (FFPE) samples.
To address the desire to perform RNA-Seq on FFPE materials, researchers from the University of North Carolina at Chapel Hill evaluated two different library preparation protocols that could be compatible for use with small RNA fragments. They obtained paired Fresh Frozen (FF) and FFPE RNAs from multiple tumors and subjected these to different gene expression profiling methods. They tested 11 human breast tumor samples using: (a) FF RNAs by microarray, mRNA-Seq, Ribo-Zero-Seq and DSN-Seq (Duplex-Specific Nuclease) and (b) FFPE RNAs by Ribo-Zero-Seq and DSN-Seq. They also performed these different RNA-Seq protocols using 10 TCGA tumors as a validation set. The data from paired RNA samples showed high concordance in transcript quantification across all protocols and between FF and FFPE RNAs. In both FF and FFPE, Ribo-Zero-Seq removed rRNA with comparable efficiency as mRNA-Seq, and it provided an equivalent or less biased coverage on gene 3[prime] ends. Compared to mRNA-Seq where 69% of bases were mapped to the transcriptome, DSN-Seq and Ribo-Zero-Seq contained significantly fewer reads mapping to the transcriptome (20-30%); in these RNA-Seq protocols, many if not most reads mapped to intronic regions. Approximately 14 million reads in mRNA-Seq and 45-65 million reads in Ribo-Zero-Seq or DSN-Seq were required to achieve the same gene detection levels as a standard Agilent DNA microarray.