Aug 31, 2023

Public workspaceQaigen RNEasy RNA extration protocol V.2

DOI
dx.doi.org/10.17504/protocols.io.dm6gp337dvzp/v2
  • 1University of Western Ontario;
  • 2Western University
Michael Dan Siemon
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DOI: dx.doi.org/10.17504/protocols.io.dm6gp337dvzp/v2
External link: https://www.qiagen.com/us/resources/download.aspx?id=c80685c0-4103-49ea-aa72-8989420e3018&lang=en
Protocol Citation: Jessica Pardy, Michael Dan Siemon, Dilan Joseph, Justin Donovan, Richard Gibson, christopher.degroot, Amanda Hamilton 2023. Qaigen RNEasy RNA extration protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp337dvzp/v2Version created by Michael Dan Siemon
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
This protocol was used for RNA extraction of wastewater samples for wastewater-based epidemiology at Western University.
Created: August 31, 2023
Last Modified: August 31, 2023
Protocol Integer ID: 87217
Keywords: wastewater-based epidemiology, Qiagen, RNA extraction, ImPaKT
Abstract
This protocol was employed by Western University for RNA extraction of wastewater samples for wastewater-based epimelogy in London, Ontario, Canada and surrounding area. The protocol was adapted from QIAamp® Viral RNA Mini Handbook for use with the Qiagen RNeasy extraction kit.
Materials
This protocol requires QIAamp RNeasy viral minikit - Qiagen
Protocol modified from QIAamp® Viral RNA Mini Handbook
Protocol modified from QIAamp® Viral RNA Mini Handbook
Thoroughly mix wastewater sample then aliquot 40 mL into 50 mL Falcon tube. Centrifuge at 12000 RPM for 90 min. Decant supernatant, assume 280 µl pellet.
Pipet 1120 µl prepared Buffer AVL into Falcon tube with sample
Pulse-vortex samples for 15s every 2 minutes for 8 minutes
Add 1120ul ethanol then centrifuge at 4000RPM for 5 minutes
Process supernatant through QiAmp Mini column by adding 750 µl and centrifuging at 8000 rpm for 1 min. Remove flow-through and repeat until all supernatant has been processed
Add 500 µl Buffer AW1 to QiAmp Mini column and centrifuge at 6000 x g (8000 rpm) for 1 min. Replace the collection tube with a clean 2 ml collection tube
Add 500 µl Buffer AW2 to QiAmp Mini column and centrifuge at 6000 x g (8000 rpm) for 1 min. Replace the collection tube with a clean 2 ml collection tube
Dry membrane by centrifuging at 13000 rpm for 3 min
Place the QIAamp Mini column in a clean 1.5 ml eppendorf tube. Add 60 µl Buffer AVE equilibrated to room temperature.
Centrifuge at 6000 x g (8000 rpm) for 1 min
Protocol references
Adapted from:
QIAamp Viral RNA Mini Handbook—QIAGEN. (2020.). Retrieved August 24, 2023, from https://www.qiagen.com/us/resources/resourcedetail?id=c80685c0-4103-49ea-aa72-8989420e3018&lang=en