Journal
SCIENTIFIC REPORTS
Volume 4, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/srep04659
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Funding
- Division of Microbiology and Infectious Diseases (DMID, NIAID, NIH, USA) [224-10-1018]
- Oak Ridge Institute for Science and Education Fellowship through DMID
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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 degrees C incubation for 1-2 hours, in cell-lysate samples either freshly prepared or previously stored frozen at -80 degrees C.
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