Tuesday, August 28, 2012

Whole-transcriptome RNA-Seq analysis from archival FFPE tumor samples (Part 2)

(See Part 1)

A large archive of FFPE tumor samples exists globally; many are readily cross-referenced to clinical records. A wealth of information is thus potentially available to search for biomarkers among expressed genes using whole-transcriptome profiling. However, RNA from FFPE samples is generally severely degraded and, in the absence of enrichment methods, rRNA reads account for a large portion of the deep sequencing information.

Epicentre's Ribo-Zero™ kits provide a reliable method to deplete rRNA from FFPE samples, enabling whole-transcriptome RNA-Seq. A recent report by Morlan et al. describes another method, called Selective Depletion of abundant RNA Species (SDRNA): the use of DNA probes homologous to human cytoplasmic and mitochondrial rRNAs.  RNA was purified using the MasterPure™ RNA Purification Kit, treated with either SDRNA or poly(A) enrichment, libraries were prepared using the Illumina® mRNA-Seq Kit or Epicentre's ScriptSeq™ mRNA-Seq Kit (now replaced by the ScriptSeq v2 Kit), and sequenced on the GAII and HiSeq 2000 instruments.

Libraries from SDRNA exhibited <1% rRNA content in uniquely mapped reads. When SDRNA libraries were compared to a poly(A)+ library, the outlier sequences were primarily identified as snRNAs, snoRNAs, scRNAs, and mirRNAs, demonstrating that a SDRNA library better represents the whole transcriptome than a poly(A)+ library. The authors mention that the Ribo-Zero Gold Kit is the only commercially available method that can effectively deplete rRNA from fragmented total RNA, and produced very comparable results to the method described in the article "with respect to the magnitude of depletion and hands-on time."

ResearchBlogging.orgMorlan JD et al. (2012). Selective Depletion of rRNA Enables Whole Transcriptome Profiling of Archival Fixed Tissue. PloS one, 7 (8) PMID: 22900061

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