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

Content Provider	PubMed Central
Author	Shatzkes, Kenneth Belete, Teferedegne Murata, Haruhiko
Copyright Year	2014
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.
Related Links	http://dx.doi.org/10.1038/srep04659
Starting Page	4659
File Format	PDF
ISSN	20452322
e-ISSN	20452322
Journal	Scientific Reports
Volume Number	4
Language	English
Publisher	Nature Publishing Group
Publisher Date	2014-04-01
Access Restriction	Open
Rights Holder	Nature Publishing Group
Subject Keyword	Science and technology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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