Qaigen RNEasy RNA extration protocol
Jessica Pardy, Michael Dan Siemon, Dilan Joseph, Justin Donovan, Richard Gibson, christopher.degroot, Amanda Hamilton
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.
Steps
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
