Dec 02, 2021

Public workspaceRNA Extraction from Wastewater Concentrates Using RNeasy and Zymo Kits

  • 1Method Developer
  • GenomeTrakr
  • APHL Wastewater Surveillance Community of Practice
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Protocol CitationJacquelina.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 23, 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)
  1. 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)
  2. Adjustable Calibrated Micropipettors (0.2 – 1000 µl), dedicated for RNA work only.
  3. Filter barrier aerosol resistant micropipettor tips DNase/RNase free (0.2 – 1000 µl)
  4. Hype-Wipe Disinfecting Towelettes (Fisher Scientific 14-412-56 or equivalent)
  5. 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)
  6. Qiagen RNeasy Mini Kit (Qiagen 74104) OR
  7. OneStep™ PCR Inhibitor Removal Kit (Zymo Research, D6030)

B. EQUIPMENT AND SUPPLIES (Midi KIt)
  1. 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)
  2. Adjustable Calibrated Micropipettors (0.2 – 1000 µl), dedicated for RNA work only.
  3. Filter barrier aerosol resistant micropipettor tips DNase/RNase free (0.2 – 1000 µl)
  4. Hype-Wipe Disinfecting Towelettes (Fisher Scientific 14-412-56 or equivalent)
  5. 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)
  6. 5 ml low bind centrifuge tubes (Eppendorf 0030122348)
  7. 5 ml Stripettes (Fisher Scientific 07-200-9, or equivalent)
  8. Pipet Boy ACU (Integra Biosciences 155000, or equivalent)
  9. 15 ml conical tubes (Fisher Scientific 14-959-49D, or equivalent)
  10. Qiagen RNeasy Midi Kit (Qiagen 75144)
  11. OneStep™ PCR Inhibitor Removal Kit (Zymo Research, D6030)

C. REAGENTS
  1. ReagentGuanidine IsothiocyanateThermo FisherCatalog #15535016 , or equivalent
  2. ReagentNuclease-Free WaterThermo Fisher ScientificCatalog #AM9937 , or equivalent
3. ReagentEthanol, 200 proofSigma AldrichCatalog #E7023 , or equivalent
4. ReagentTris (1 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9856 , or equivalent
5. ReagentEDTA (0.5 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9261 , or equivalent
Protocol materials
ReagentTris (1 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9856
ReagentEDTA (0.5 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9261
ReagentGuanidine IsothiocyanateThermo FisherCatalog #15535016
ReagentNuclease-Free WaterThermo Fisher ScientificCatalog #AM9937
ReagentEthanol, 200 proofMerck MilliporeSigma (Sigma-Aldrich)Catalog #E7023
ReagentChloroform Merck MilliporeSigma (Sigma-Aldrich)Catalog #288306
Before start
Heat Primer TE at Temperature70 °C for at least Duration00:10:00 prior to use.
Protocol
Primer TE (10mM Tris, 0.1mM EDTA, pH8.0)
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 Amount500 µL 6M GITC.
Protocol
6M GITC
NAME
6M GITC
CREATED BY
Jessica L Jones
.

Aseptically mix Amount7.09 g ReagentGuanidine IsothiocyanateThermo FisherCatalog #15535016 in sterile distilled or ultrapure water to Amount4.5 mL

Bring total volume up to Amount10 mL with additional sterile distilled or ultrapure water

Store at TemperatureRoom temperature in the dark (or in light occluding tube)

Note
Solution is only stable for 1 month after preparation.

Vortex Duration00:01:15 +/- 15 sec to dissolve concentrate.


1m 15s
Add Amount700 µL of 50% EtOH and invert twice.

Pipette Amount700 µL of sample onto a RNeasy mini spin column.

Centrifuge Centrifigation10000 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 Centrifigation10000 x g, 00:01:00 .

1m
Place column in new collection tube and discard flow through.
Add Amount700 µL RW1 buffer to spin column and incubate for Duration00:15:00 at room temperature.

15m
Centrifuge Centrifigation10000 x g, 00:01:00 .
1m
Place column in new collection tube and discard flow through.
Add Amount500 µL RPE to spin column and incubate for Duration00:15:00 .

15m
Centrifuge Centrifigation10000 x g, 00:01:00 .
1m
Add an additional Amount500 µL of RPE to Mini spin column.

Note
Incubation is not required at this step.

Centrifuge at maximum speed Centrifigation>16000 x g, 00:02:00 .

Note
Maximum speed should be ≥16,000 x g.

2m
Transfer column to new collection tube.
Centrifuge Centrifigation16000 x g, 00:01:00 .
1m
Carefully transfer column to 1.5 ml low-retention/siliconized RNase/DNase free microcentrifuge tube.
Pipette Amount50 µL pre-heated (70°C) Primer TE onto silica-gel membrane of column.
Protocol
Primer TE (10mM Tris, 0.1mM EDTA, pH8.0)
NAME

Primer TE (10mM Tris, 0.1mM EDTA, pH8.0)

CREATED BY
Jessica L Jones


Mix components together.
Amount100 µL ReagentTris (1 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9856,, or equivalent.

Amount20 µL ReagentEDTA (0.5 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9261,, or equivalent.

Amount9.88 mL ReagentNuclease-Free WaterThermo Fisher ScientificCatalog #AM9937 , or equivalent.

Store at TemperatureRoom temperature .

Centrifuge Centrifigation10000 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 Amount50 µL pre-heated (70°C) Primer TE onto silica-gel membrane of column.
Pipette the eluted RNA (from Step 22) Amount50 µL back onto column.
Centrifuge Centrifigation10000 x g, Room temperature, 00:01:00 .
1m
Discard column and place tube with RNA on TemperatureOn ice to prepare Zymo column.

Prepare Zymo column per manufacturer's instructions.
Insert column into a collection tube.
Open the cap and add Amount600 µL of Prep-solution.

Centrifuge at Centrifigation8000 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 Centrifigation8000 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 Temperature-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 Amount400 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.

Optional
Add Amount800 µL of ReagentChloroform Sigma AldrichCatalog #288306..
Vortex Duration00:00:45 +/- 15 sec .
45s
Centrifuge Centrifigation3000 x g, Room temperature, 00:05:00 .
5m
Transfer aqueous layer to 5 ml Eppendorf low bind centrifuge tube.
Add Amount2 mL 6M GITC.
Protocol
6M GITC
NAME
6M GITC
CREATED BY
Jessica L Jones

Aseptically mix Amount7.09 g ReagentGuanidine IsothiocyanateThermo FisherCatalog #15535016 in sterile distilled or ultrapure water to Amount4.5 mL

Bring total volume up to Amount10 mL with additional sterile distilled or ultrapure water

Store at TemperatureRoom temperature in the dark (or in light occluding tube)

Note
Solution is only stable for 1 month after preparation.

Vortex Duration00:01:15 +/- 15 sec .

1m 15s
Add Amount2.8 mL of 50% EtOH and invert twice.

PipetteAmount4 mL of sample onto a RNeasy Midi spin column.

Centrifuge Centrifigation5000 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 Centrifigation5000 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.
AddAmount4 mL RW1 buffer to Midi spin column.

Incubate for Duration00:15:00 atTemperatureRoom temperature .

15m
Centrifuge Centrifigation5000 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 Amount4 mL RPE to Midi spin column and incubate for Duration00:15:00 at TemperatureRoom temperature .

15m
Centrifuge Centrifigation5000 x g, Room temperature, 00:05:00 .
5m
Transfer column to new collection tube and add an additional Amount4 mL of RPE to the Midi spin column.

Note
Incubation not required at this step.

Centrifuge Centrifigation5000 x g, Room temperature, 00:10:00 .
10m
Carefully transfer column to a sterile, nuclease-free 15 ml conical tube.
Pipette Amount150 µL heated (Temperature70 °C ) Primer TE onto silica-gel membrane of Midi column.

Incubate forDuration00:01:00 at TemperatureRoom temperature .

1m
Centrifuge Centrifigation5000 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 Amount150 µL of heated Primer TE onto the Midi column.

Pipette the eluted RNA (Step 55) back onto column (~Amount150 µL ).

Centrifuge Centrifigation5000 x g, Room temperature, 00:03:00 .
3m
Discard column and place tube with RNA TemperatureOn 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 Amount600 µL of Prep-solution.
Centrifuge at Centrifigation8000 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 (~Amount150 µL each) .

Centrifuge Centrifigation8000 x g, Room temperature, 00:03:00 .
3m
Combine RNA from columns into one fresh low-retention/siliconized RNase/DNase free micriocentrifuge tube.