Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae

Abstract

Accurate genomic sequencing demands high-quality double-stranded RNA (dsRNA). Existing methods for dsRNA extraction from yeast, fungi, and plants primarily rely on cellulose, suitable only for small volume extractions, or the time-consuming lithium chloride precipitation. To streamline the traditional phenol-chloroform-based dsRNA extraction method, the main challenge is the reduction of mitochondrial DNA (mtDNA) and Single Stranded RNA (ssRNA) to no detectable levels after gel electrophoresis. This challenge is successfully addressed through the modified approach described here, involving phenol extraction at low pH, followed by the addition of ammonium sulfate to the aqueous buffer. The dsRNA isolated using this novel method exhibits comparable quality to that obtained through cellulose purification, and it is readily amenable to RT-PCR. Moreover, a single batch of yeast cell RNA isolation requires only 2-3 h of hands-on time, thus simplifying and expediting the process significantly. • Buffers were redesigned from [32,33,35]. • No DNASE, Ribonuclease A or beads were used during the purification. • Simple and inexpensive dsRNA extraction and purification method is described.

Keywords

• Viral dsRNA
• Phenol extraction
• Ammonium sulphate
• RT-PCR
• Sequencing