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

Scientific reports (2014-04-12)
Kenneth Shatzkes, Belete Teferedegne, Haruhiko Murata
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
IGEPAL® CA-630, for molecular biology
Sigma-Aldrich
Trizma® hydrochloride solution, pH 7.4, BioPerformance Certified, 1 M, suitable for cell culture