1 research outputs found
Simple, Inexpensive RNA Isolation and OneâStep RTâqPCR Methods for SARSâCoVâ2 Detection and General Use
The most common method for RNA detection involves reverse transcription followed by quantitative polymerase chain reaction (RTâqPCR) analysis. Commercial oneâstep master mixesâwhich include both a reverse transcriptase and a thermostable polymerase and thus allow performing both the RT and qPCR steps consecutively in a sealed wellâare key reagents for SARSâCoVâ2 diagnostic testing; yet, these are typically expensive and have been affected by supply shortages in periods of high demand. As an alternative, we describe here how to express and purify Taq polymerase and MâMLV reverse transcriptase and assemble a homemade oneâstep RTâqPCR master mix. This mix can be easily assembled from scratch in any laboratory equipped for protein purification. We also describe two simple alternative methods to prepare clinical swab samples for SARSâCoVâ2 RNA detection by RTâqPCR: heatâinactivation for direct addition, and concentration of RNA by isopropanol precipitation. Finally, we describe how to perform RTâqPCR using the homemade master mix, how to prepare in vitroâtranscribed RNA standards, and how to use a fluorescence imager for endpoint detection of RTâPCR amplification in the absence of a qPCR machine In addition to being useful for diagnostics, these versatile protocols may be adapted for nucleic acid quantification in basic research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of a oneâstep RTâqPCR master mix using homemade enzymes Basic Protocol 2: Preparation of swab samples for direct RTâPCR Alternate Protocol 1: Concentration of RNA from swab samples by isopropanol precipitation Basic Protocol 3: Oneâstep RTâqPCR of RNA samples using a realâtime thermocycler Support Protocol: Preparation of RNA concentration standards by in vitro transcription Alternate Protocol 2: Oneâstep RTâPCR using endpoint fluorescence detectio