RNA Extraction from Wastewater Concentrates Using RNeasy and Zymo Kits
Jacquelina.Woods, rachel.rodriguez
Abstract
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method describes the extraction of RNA from viral concentrates using the RNeasy and Zymo kits.
Before start
Heat Primer TE at 70°C for at least 0h 10m 0s prior to use.
Primer TE (10mM Tris, 0.1mM EDTA, pH8.0)
Steps
RNeasy Mini
Obtain one virus concentrate (if concentrate is frozen, allow thawing) from Virus Concentration from Wastewater Using PEG Precipitation and Ultracentrifugation (protocols.io)
Add 500µL 6M GITC.
Vortex 0h 1m 15s to dissolve concentrate.
Add 700µL of 50% EtOH and invert twice.
Pipette 700µLof sample onto a RNeasy mini spin column.
Centrifuge 10000x g.
Place column in new collection tube and discard flow through.
Add remaining sample from Step 4 to column.
Centrifuge 10000x g.
Place column in new collection tube and discard flow through.
Add 700µL RW1 buffer to spin column and incubate for 0h 15m 0s at room temperature.
Centrifuge 10000x g.
Place column in new collection tube and discard flow through.
Add 500µL RPE to spin column and incubate for 0h 15m 0s.
Centrifuge 10000x g.
Add an additional 500µL of RPE to Mini spin column.
Centrifuge at maximum speed >16000x g.
Transfer column to new collection tube.
Centrifuge 16000x g.
Carefully transfer column to 1.5 ml low-retention/siliconized RNase/DNase free microcentrifuge tube.
Pipette 50µL pre-heated (70°C) Primer TE onto silica-gel membrane of column.
Centrifuge 10000x g.
Pipette an additional 50µL pre-heated (70°C) Primer TE onto silica-gel membrane of column.
Pipette the eluted RNA (from Step 22) 50µL back onto column.
Centrifuge 10000x g.
Discard column and place tube with RNA on On iceto prepare Zymo column.
Prepare Zymo column per manufacturer's instructions.
Insert column into a collection tube.
Open the cap and add 600µL of Prep-solution.
Centrifuge at 8000x g.
Transfer Zymo column into a clean 1.5 or 2.0 ml low-retention/siliconized RNase/DNase free microcentrifuge tube.
Transfer RNA from Step 26 to prepared Zymo One Step RT-PCR inhibitor remover column.
Centrifuge 8000x g.
Discard column and save RNA in the 1.5 or 2.0 ml low-retention/siliconized RNase/DNase free microcentrifuge tube.
Proceed with RT-qPCR or store RNA at -70°C.
RNeasy Midi
Obtain two virus concentrates (if concentrate is frozen, allow thawing) from Virus Concentration from Wastewater Using PEG Precipitation and Ultracentrifugation (protocols.io)
Optional chloroform clean-up.
Add 800µLof
Vortex 0h 0m 45s.
Centrifuge 3000x g.
Transfer aqueous layer to 5 ml Eppendorf low bind centrifuge tube.
Add 2mL 6M GITC.
Vortex 0h 1m 15s.
Add 2.8mL of 50% EtOH and invert twice.
Pipette4mL of sample onto a RNeasy Midi spin column.
Centrifuge 5000x g.
Discard flow through and return Midi column to tube.
Add remaining sample (from Step 40) to Midi column.
Centrifuge 5000x g.
Place column in new collection tube (sterile, nuclease-free 15 ml conical tube) and discard flow through.
Add4mL RW1 buffer to Midi spin column.
Incubate for 0h 15m 0s atRoom temperature.
Centrifuge 5000x g.
Place column in new collection tube (sterile, nuclease-free 15 ml conical tube) and discard flow through.
Add 4mL RPE to Midi spin column and incubate for 0h 15m 0s at Room temperature.
Centrifuge 5000x g.
Transfer column to new collection tube and add an additional 4mLof RPE to the Midi spin column.
Centrifuge 5000x g.
Carefully transfer column to a sterile, nuclease-free 15 ml conical tube.
Pipette 150µLheated (70°C) Primer TE onto silica-gel membrane of Midi column.
Incubate for0h 1m 0s at 70Room temperature.
Centrifuge 5000x g.
Add an additional 150µLof heated Primer TE onto the Midi column.
Pipette the eluted RNA (Step 55) back onto column (~150µL).
Centrifuge 5000x g.
Discard column and place tube with RNA On ice to prepare Zymo column.
Prepare two (2) Zymo One Step RT-PCR inhibitor remover columns per manufacturer's instructions.
Insert Zymo column into a collection tube.
Open the cap and add 600µL of Prep-solution.
Centrifuge at 8000x g.
Transfer Zymo column into a clean 1.5 or 2.0 ml low-retention/siliconized RNase/DNase free micriocentrifuge tube.
Transfer RNA from Step 59 to prepared Zymo One Step RT-PCR inhibitor remover columns (~150µLeach) .
Centrifuge 8000x g.
Combine RNA from columns into one fresh low-retention/siliconized RNase/DNase free micriocentrifuge tube.
Proceed with RT-qPCR (RT-qPCR Detection of Process Controls (Murine noroviurs and crAssphage) from Wastewater (protocols.io) and RT-qPCR Detection of SARS-CoV-2 from Wastewater Using the AB 7500 (protocols.io)) or store at-70°C .
