A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR
書誌事項
- 公開日
- 2014-04-11
- 権利情報
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- https://creativecommons.org/licenses/by/3.0/
- https://creativecommons.org/licenses/by/3.0/
- DOI
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- 10.1038/srep04659
- 公開者
- Springer Science and Business Media LLC
説明
<jats:title>Abstract</jats:title><jats:p>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.</jats:p>
収録刊行物
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- Scientific Reports
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Scientific Reports 4 (1), 2014-04-11
Springer Science and Business Media LLC