Unearthing archival tumor RNA

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Science Translational Medicine  19 Aug 2015:
Vol. 7, Issue 301, pp. 301ec142
DOI: 10.1126/scitranslmed.aad0230

Large-scale next-generation sequencing efforts of recent years have defined the somatic DNA changes that underpin many different types of human cancer. In parallel, tumor RNA has been widely studied through sequencing to provide gene expression profiles as well as the base-pair resolution of transcribed mutations. A major challenge of studying tumor RNA is the difficulty of obtaining a high-quality, undegraded sample—especially RNA that has been fixed and preserved by formalin and paraffin, as is routine in clinical practice. Now, Cieslik et al. describe an important RNA sequencing method that enables high-quality sequencing from real life–partially degraded tumor RNA.

A consequence of RNA degradation is that the enrichment of transcripts becomes increasingly inefficient. For example, one principal method for transcript enrichment that targets the poly-A tails of messenger RNA (mRNA) becomes effectively impossible when these tails are degraded. Therefore, outside of research settings with dedicated practices for preserving tumor RNA through rapid freezing of specimens, high-quality RNA sequences are difficult to generate. Two current methods for enriching mRNA transcripts from total RNA samples rely on either targeting poly-A tails or depleting ribosomal RNA (rRNA). In the new work, the authors targeted complementary DNA of mRNA gene transcripts directly through exome-capture technology, which uses oligonucleotides (“baits”) aimed at exons to extract genes. Cieslik et al. demonstrated that exome-capture RNA sequencing generated data of a quality that was comparable with standard RNA sequencing protocols. In fact, with respect to mutation calling and fusion-gene discovery, exome-capture RNA sequencing was superior to standard protocols, probably because relevant transcript were more efficiently enriched. The authors demonstrated that exome-capture RNA sequencing performed well not only for ideal RNA from fresh frozen tissue, but also in artificially degraded RNA and degraded RNA from formalin-fixed, paraffin-embedded clinical specimens.

The new RNA sequencing protocol is likely to greatly enhance the feasibility of tumor RNA studies, both in research and clinical settings. A possible limitation of their method is the exclusion of noncoding RNA, which is not explicitly targeted by baits. However, particularly in clinical practice, in which protein-coding transcript are the major RNA species of interest, exome-capture RNA sequencing may justifiably become the method of choice.

M. Cieslik et al., The use of exome capture RNA-seq for highly degraded RNA with application to clinical cancer sequencing. Genome Res. 10.1101/gr.189621.115 (2015). [Abstract]

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