Abstract 4857: Improved technology for ribosomal RNA (rRNA) removal from formalin-fixed paraffin-embedded (FFPE) total RNA

Abstract

Abstract Deep, massively parallel sequencing of cDNA generated from RNA (“RNA-Seq”) is rapidly gaining momentum for transcript profiling, discovery of novel transcripts, and identification of alternative splicing events. Current methods for making sequencer-specific di-tagged DNA fragment libraries for RNA-Seq typically comprise first, preparing rRNA-depleted RNA from total RNA samples that are usually of good quality followed by the synthesis of the di-tagged cDNA sequencing templates. In the past few years however, it has also become evident from microarray and qPCR studies that formalin-fixed paraffin-embedded (FFPE) cancer tissues hold valuable secrets about diseased states. However, RNA-Seq libraries prepared from such highly fragmented FFPE RNA cancer samples yield limited information since these libraries contain a majority of rRNA reads, which decreases sequencing depth and coverage. Current commercially available rRNA-removal kits are not designed to remove fragments of rRNA, which poses a significant limitation for preparing highly informative RNA-Seq libraries from FFPE RNA samples. Here, we present RNA-Seq results obtained using a novel rRNA removal technology (“Ribo-ZERO™”) and a novel, ligation-free process for preparing directional di-tagged DNA fragment libraries (“ScriptSeq™ Technology”) for RNA-Seq. Using these methods, directional di-tagged DNA fragment libraries can be prepared in about 6 hours from either intact or fragmented (e.g., FFPE) total RNA samples (as little as 100 ng FFPE total RNA sample required). Less than 2 % of the sequence reads from libraries generated from total RNA from either intact or FFPE samples map to rRNA sequences (28S, 18S, 5.8S and 5S). This reduction in rRNA sequence reads from FFPE RNA samples improves sequence depth and coverage, and increases the percentage of uniquely mapped reads, increasing the information obtained from these diseased samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4857. doi:10.1158/1538-7445.AM2011-4857