A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR.
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.
1] Laboratory of DNA Viruses, Division of Viral Products, OVRR, CBER, FDA, Bethesda, MD 20892, USA [2] Laboratory of Pediatric and Respiratory Viral Diseases, Division of Viral Products, OVRR, CBER, FDA, Bethesda, MD 20892, USA [3] [4].
Kenneth
K
Shatzkes
1] Laboratory of DNA Viruses, Division of Viral Products, OVRR, CBER, FDA, Bethesda, MD 20892, USA [2].
Belete
B
Teferedegne
Laboratory of DNA Viruses, Division of Viral Products, OVRR, CBER, FDA, Bethesda, MD 20892, USA.
Haruhiko
H
Murata
D000818:Animals; D004285:Dogs; D009981:Influenza B virus / Q000235:genetics; D061985:Madin Darby Canine Kidney Cells; D046210:Microfluidic Analytical Techniques; D012313:RNA / Q000032:analysis* / Q000378:metabolism; D020871:RNA Stability; D012367:RNA, Viral / Q000032:analysis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*
D012367:RNA, Viral; D012313:RNA