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

• Published in: Scientific reports Vol. 4; no. 1; p. 4659
• Main Authors: Shatzkes, Kenneth, Teferedegne, Belete, Murata, Haruhiko
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 11.04.2014; Nature Publishing Group
• Subjects: 38/47; 38/56; 38/62; 38/77; 38/88; 38/90; 631/1647/2017/2003; 631/1647/2234; 631/326/596/1578; 631/337/2019; Animals; Dogs; Experiments; Humanities and Social Sciences; Infections; Influenza; Influenza B virus - genetics; Lysates; Madin Darby Canine Kidney Cells; Microfluidic Analytical Techniques; multidisciplinary; Polymerase chain reaction; Purification; Reagents; Reverse Transcriptase Polymerase Chain Reaction - economics; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse transcription; Ribonucleic acid; RNA; RNA - analysis; RNA - metabolism; RNA Stability; RNA, Viral - analysis; RNA, Viral - metabolism; Science; Standard deviation; Viruses
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep04659

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.