Isolating Viral and Host RNA Sequences from Archival Material and Production of cDNA Libraries for High-Throughput DNA Sequencing

Abstract

The vast majority of surgical biopsy and post-mortem tissue samples are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. As an example, the viral RNA genome of the 1918 pandemic influenza A virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Using the protocols described here, the full genome of the 1918 virus was determined at high coverage in one high-throughput sequencing run of a cDNA library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. This basic methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of infectious diseases.

Keywords: RNA; cDNA; high-throughput sequencing; influenza; library; polymerase chain reaction.

Figure 1

Typical FFPE total RNA on Bioanalyer (The small peak in front is the RNA lower marker).

Figure 2

Typical sequencing library on Bioanalyer (The major peak at ~300bp is the library peak. The small peak in front of and another small peak behind the major peak are the lower and higher marker peaks).