Dec 02, 2021
RNA Extraction from Wastewater Concentrates Using RNeasy and Zymo Kits
- 1Method Developer
- GenomeTrakr
- APHL Wastewater Surveillance Community of Practice
Protocol Citation: Jacquelina.Woods, rachel.rodriguez 2021. RNA Extraction from Wastewater Concentrates Using RNeasy and Zymo Kits. protocols.io https://dx.doi.org/10.17504/protocols.io.bygvptw6
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
We use this protocol and it’s working
Created: September 22, 2021
Last Modified: December 02, 2021
Protocol Integer ID: 53493
Keywords: GenomeTrakr, wastewater, SARS-CoV-2, RNA, Extraction, RNeasy, Zymo
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.
Materials
A. EQUIPMENT AND SUPPLIES (Mini KIt)
- Centrifuge capable of speeds of ≥16,000 x g (e.g, Eppendorf 5424 or 5415 D), and with rotors capable of holding 1.5ml and 2.0ml microcentrifuge tubes (Eppendorf 2231000655 or equivalent)
- Adjustable Calibrated Micropipettors (0.2 – 1000 µl), dedicated for RNA work only.
- Filter barrier aerosol resistant micropipettor tips DNase/RNase free (0.2 – 1000 µl)
- Hype-Wipe Disinfecting Towelettes (Fisher Scientific 14-412-56 or equivalent)
- DNase/RNase-free microcentrifuge tubes 1.5 mL, non-stick, low retention, siliconized (Life Technologies AM12450) OR DNase/RNase-free microcentrifuge tubes 2.0 mL, non-stick, low retention, siliconized (Life Technologies AM12475)
- Qiagen RNeasy Mini Kit (Qiagen 74104) OR
- OneStep™ PCR Inhibitor Removal Kit (Zymo Research, D6030)
B. EQUIPMENT AND SUPPLIES (Midi KIt)
- Centrifuge capable of speeds of ≥8,000 x g (e.g, ThermoFisher RC6), and with rotors capable of holding 15ml tubes (ThermoFisher Scientific, 36-101-0816, or equivalent)
- Adjustable Calibrated Micropipettors (0.2 – 1000 µl), dedicated for RNA work only.
- Filter barrier aerosol resistant micropipettor tips DNase/RNase free (0.2 – 1000 µl)
- Hype-Wipe Disinfecting Towelettes (Fisher Scientific 14-412-56 or equivalent)
- DNase/RNase-free microcentrifuge tubes 1.5 mL, non-stick, low retention, siliconized (Life Technologies AM12450) OR DNase/RNase-free microcentrifuge tubes 2.0 mL, non-stick, low retention, siliconized (Life Technologies AM12475)
- 5 ml low bind centrifuge tubes (Eppendorf 0030122348)
- 5 ml Stripettes (Fisher Scientific 07-200-9, or equivalent)
- Pipet Boy ACU (Integra Biosciences 155000, or equivalent)
- 15 ml conical tubes (Fisher Scientific 14-959-49D, or equivalent)
- Qiagen RNeasy Midi Kit (Qiagen 75144)
- OneStep™ PCR Inhibitor Removal Kit (Zymo Research, D6030)
C. REAGENTS
- Guanidine IsothiocyanateThermo FisherCatalog #15535016 , or equivalent
- Nuclease-Free WaterThermo Fisher ScientificCatalog #AM9937 , or equivalent
3. Ethanol, 200 proofSigma AldrichCatalog #E7023 , or equivalent
4. Tris (1 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9856 , or equivalent
5. EDTA (0.5 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9261 , or equivalent
Protocol materials
Tris (1 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9856
EDTA (0.5 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9261
Guanidine IsothiocyanateThermo FisherCatalog #15535016
Nuclease-Free WaterThermo Fisher ScientificCatalog #AM9937
Ethanol, 200 proofMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
Chloroform Merck MilliporeSigma (Sigma-Aldrich)Catalog #288306
Before start
Heat Primer TE at 70 °C for at least 00:10:00 prior to use.
Protocol
NAME
Primer TE (10mM Tris, 0.1mM EDTA, pH8.0)
CREATED BY
Jessica L Jones
Safety information
This document contains two separate protocols.
If RNA concentrations are expected to be low, the RNA Mini column should be used (follow Section 1). If RNA concentrations are expected to be high, the RNA Midi column is recommended (follow Section 2).
RNeasy Mini
RNeasy Mini
43m 15s
43m 15s
Obtain one virus concentrate (if concentrate is frozen, allow thawing) from Virus Concentration from Wastewater Using PEG Precipitation and Ultracentrifugation (protocols.io)
Add 500 µL 6M GITC.
Protocol
NAME
6M GITCCREATED BY
Jessica L Jones
.
Aseptically mix 7.09 g Guanidine IsothiocyanateThermo FisherCatalog #15535016 in sterile distilled or ultrapure water to 4.5 mL
Bring total volume up to 10 mL with additional sterile distilled or ultrapure water
Store at Room temperature in the dark (or in light occluding tube)
Note
Solution is only stable for 1 month after preparation.
Vortex 00:01:15 +/- 15 sec to dissolve concentrate.
1m 15s
Add 700 µL of 50% EtOH and invert twice.
Pipette 700 µL of sample onto a RNeasy mini spin column.
Centrifuge 10000 x g, 00:01:00 .
1m
Place column in new collection tube and discard flow through.
Add remaining sample from Step 4 to column.
Centrifuge 10000 x g, 00:01:00 .
1m
Place column in new collection tube and discard flow through.
Add 700 µL RW1 buffer to spin column and incubate for 00:15:00 at room temperature.
15m
Centrifuge 10000 x g, 00:01:00 .
1m
Place column in new collection tube and discard flow through.
Add 500 µL RPE to spin column and incubate for 00:15:00 .
15m
Centrifuge 10000 x g, 00:01:00 .
1m
Add an additional 500 µL of RPE to Mini spin column.
Note
Incubation is not required at this step.
Centrifuge at maximum speed >16000 x g, 00:02:00 .
Note
Maximum speed should be ≥16,000 x g.
2m
Transfer column to new collection tube.
Centrifuge 16000 x g, 00:01:00 .
1m
Carefully transfer column to 1.5 ml low-retention/siliconized RNase/DNase free microcentrifuge tube.
Pipette 50 µL pre-heated (70°C) Primer TE onto silica-gel membrane of column.
Protocol
NAME
Primer TE (10mM Tris, 0.1mM EDTA, pH8.0)
CREATED BY
Jessica L Jones
Mix components together.
100 µL Tris (1 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9856,, or equivalent.
20 µL EDTA (0.5 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9261,, or equivalent.
9.88 mL Nuclease-Free WaterThermo Fisher ScientificCatalog #AM9937 , or equivalent.
Store at Room temperature .
Centrifuge 10000 x g, 00:01:00 .
Note
Material that passed through column contains the viral RNA being isolated and is in the 1.5 ml low-retention/siliconized RNase/DNase free microcentrifuge tube in which the column was placed.
1m
Pipette an additional 50 µL pre-heated (70°C) Primer TE onto silica-gel membrane of column.
Pipette the eluted RNA (from Step 22) 50 µL back onto column.
Centrifuge 10000 x g, Room temperature, 00:01:00 .
1m
Discard column and place tube with RNA on On ice to prepare Zymo column.
Prepare Zymo column per manufacturer's instructions.
Insert column into a collection tube.
Open the cap and add 600 µL of Prep-solution.
Centrifuge at 8000 x g, Room temperature, 00:03:00 .
3m
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 8000 x g, 00:03:00 .
3m
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
RNeasy Midi
5m 45s
5m 45s
Obtain two virus concentrates (if concentrate is frozen, allow thawing) from Virus Concentration from Wastewater Using PEG Precipitation and Ultracentrifugation (protocols.io)
Note
Total volume should be approximately 400 µL .
Optional chloroform clean-up.
If high amounts of solids were present in the original wastewater sample (i.e., bottles 1 and 2), the chloroform clean-up is recommended prior to proceeding with the Midi column extraction. If few solids were present (i.e., bottles 3 and 4), this step can be omitted and the sample can be extracted starting with Step 35.
Add 800 µL of Chloroform Sigma AldrichCatalog #288306..
Vortex 00:00:45 +/- 15 sec .
45s
Centrifuge 3000 x g, Room temperature, 00:05:00 .
5m
Transfer aqueous layer to 5 ml Eppendorf low bind centrifuge tube.
Add 2 mL 6M GITC.
Protocol
NAME
6M GITCCREATED BY
Jessica L Jones
Aseptically mix 7.09 g Guanidine IsothiocyanateThermo FisherCatalog #15535016 in sterile distilled or ultrapure water to 4.5 mL
Bring total volume up to 10 mL with additional sterile distilled or ultrapure water
Store at Room temperature in the dark (or in light occluding tube)
Note
Solution is only stable for 1 month after preparation.
Vortex 00:01:15 +/- 15 sec .
1m 15s
Add 2.8 mL of 50% EtOH and invert twice.
Pipette4 mL of sample onto a RNeasy Midi spin column.
Centrifuge 5000 x g, Room temperature, 00:05:00 .
5m
Discard flow through and return Midi column to tube.
Add remaining sample (from Step 40) to Midi column.
Centrifuge 5000 x g, Room temperature, 00:05:00 .
5m
Place column in new collection tube (sterile, nuclease-free 15 ml conical tube) and discard flow through.
Add4 mL RW1 buffer to Midi spin column.
Incubate for 00:15:00 atRoom temperature .
15m
Centrifuge 5000 x g, Room temperature, 00:05:00 .
5m
Place column in new collection tube (sterile, nuclease-free 15 ml conical tube) and discard flow through.
Add 4 mL RPE to Midi spin column and incubate for 00:15:00 at Room temperature .
15m
Centrifuge 5000 x g, Room temperature, 00:05:00 .
5m
Transfer column to new collection tube and add an additional 4 mL of RPE to the Midi spin column.
Note
Incubation not required at this step.
Centrifuge 5000 x g, Room temperature, 00:10:00 .
10m
Carefully transfer column to a sterile, nuclease-free 15 ml conical tube.
Pipette 150 µL heated (70 °C ) Primer TE onto silica-gel membrane of Midi column.
Incubate for00:01:00 at Room temperature .
1m
Centrifuge 5000 x g, Room temperature, 00:03:00 .
Note
Material that passed through column contains the viral RNA being isolated and is in the sterile, nuclease-free 15 ml conical tube in which the column was placed.
3m
Add an additional 150 µL of heated Primer TE onto the Midi column.
Pipette the eluted RNA (Step 55) back onto column (~150 µL ).
Centrifuge 5000 x g, Room temperature, 00:03:00 .
3m
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 600 µL of Prep-solution.
Centrifuge at 8000 x g, Room temperature, 00:03:00 .
3m
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 (~150 µL each) .
Centrifuge 8000 x g, Room temperature, 00:03:00 .
3m
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 .