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

Kenneth Shatzkes,Belete Teferedegne,Haruhiko Murata

doi:10.1038/srep04659

Kenneth Shatzkes, Belete Teferedegne + Show 1 more

Open Access

https://doi.org/10.1038/srep04659

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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.

Highlights

Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR workflows
We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription quantitative PCR (qPCR) (RT-qPCR)
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

Summary

Introduction

Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. Q uantitative PCR (qPCR) is associated with several appealing performance features such as its sensitivity (which can allow quantification of targets approaching the limiting concentration in molecular terms) and its dynamic range (which can span several orders of magnitude) Despite these obvious advantages, full realization of the potential of qPCR has been hindered, for high-throughput applications, because sample nucleic acid purification required in a conventional workflow can often be cumbersome and rate-limiting. Virus-infected cells (in a 96-well plate format) are washed and briefly exposed to a commercially available cell-lysis reagent; the resulting cell lysates are subjected to direct analysis by one-step RT-qPCR in order to measure the expression level of a viral gene target Samples prepared in this straightforward manner require minimal effort. Avoidance of exogenous RNase inhibitor addition allows per-sample cost of generating cell lysates for RT-qPCR to be essentially negligible using our buffer

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