A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR

Sci Rep. 2014 Apr 11:4:4659. doi: 10.1038/srep04659.

Abstract

Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription qPCR (RT-qPCR). The need for cumbersome RNA purification is circumvented in our assays by making use of a commercial reagent that can easily generate crude cell lysates amenable to direct analysis by one-step RT-qPCR. In the present study, we demonstrate that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercially available reagents for the purpose of generating RT-qPCR-ready cell lysates from MDCK cells infected with influenza virus. We have found that addition of exogenous RNase inhibitor as a buffer component is not essential in order to maintain RNA integrity, even following stress at 37 °C incubation for 1-2 hours, in cell-lysate samples either freshly prepared or previously stored frozen at -80 °C.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Dogs
  • Influenza B virus / genetics
  • Madin Darby Canine Kidney Cells
  • Microfluidic Analytical Techniques
  • RNA / analysis*
  • RNA / metabolism
  • RNA Stability
  • RNA, Viral / analysis
  • RNA, Viral / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction / economics
  • Reverse Transcriptase Polymerase Chain Reaction / methods*

Substances

  • RNA, Viral
  • RNA