Jul 28, 2020

Public workspaceDirect wastewater RNA extraction via the "Milk of Silica (MoS)" method - A companion method to "Sewage, Salt, Silica and SARS-CoV-2 (4S)"

DOI
dx.doi.org/10.17504/protocols.io.biwfkfbn
  • Oscar N Whitney1,
  • Basem Al-Shayeb2,
  • Alex Crits-Cristoph3,
  • Mira Chaplin4,
  • Vinson Fan1,
  • Hannah Greenwald4,
  • Adrian Hinkle4,
  • Rose Kantor4,
  • Lauren Kennedy4,
  • Anna Maurer1,
  • Robert Tjian5,
  • Kara L. Nelson6,
  • UC Berkeley Wastewater-based epidemiology consortium6
  • 1University of California, Berkeley, Tjian & Darzacq laboratory;
  • 2University of California, Berkeley, Banfield & Doudna laboratory;
  • 3University of California, Berkeley, Banfield laboratory;
  • 4University of California, Berkeley, Nelson laboratory;
  • 5University of California, Berkeley, HHMI;
  • 6University of California, Berkeley
  • Coronavirus Method Development Community
Oscar N Whitney
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DOI: dx.doi.org/10.17504/protocols.io.biwfkfbn
Protocol Citation: Oscar N Whitney, Basem Al-Shayeb, Alex Crits-Cristoph, Mira Chaplin, Vinson Fan, Hannah Greenwald, Adrian Hinkle, Rose Kantor, Lauren Kennedy, Anna Maurer, Robert Tjian, Kara L. Nelson, UC Berkeley Wastewater-based epidemiology consortium 2020. Direct wastewater RNA extraction via the "Milk of Silica (MoS)" method - A companion method to "Sewage, Salt, Silica and SARS-CoV-2 (4S)". protocols.io https://dx.doi.org/10.17504/protocols.io.biwfkfbn
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 in our laboratory to routinely isolate RNA from wastewater samples
Created: July 23, 2020
Last Modified: July 28, 2020
Protocol Integer ID: 39591
Keywords: SARS-CoV-2, COVID19, Wastewater-based epidemiology, Direct capture, RNA extraction,
Abstract
The following protocol describes the "4S" (Sewage, Salt, Silica and SARS-CoV-2) workflow applied to using dry silica powder as an RNA-binding matrix instead of silica spin columns. This offers an even more economical alternative, requiring only centrifugation to extract RNA from wastewater. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.



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Materials
MATERIALS
ReagentTris
ReagentEDTA
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentSodium acetateMerck MilliporeCatalog #1.06268.1000
ReagentCentrifuge
ReagentTE bufferThermo Fisher Scientific
ReagentEthanol
ReagentIsopropanolMerck MilliporeCatalog #109634
ReagentSilicon dioxide ~99% 0.5-10 µm (approx. 80% between 1-5 µm)Millipore SigmaCatalog #SIGMA S5631
STEP MATERIALS
ReagentDurapore® Membrane Filter 5.0 µmMillipore SigmaCatalog #SVLP04700
ReagentSwinnex Filter HolderMillipore SigmaCatalog #SX0004700
ReagentMagnetic Funnel 300mL 47mmPallCatalog #4242
ReagentSilicon dioxide ~99% 0.5-10 µm (approx. 80% between 1-5 µm)Millipore SigmaCatalog #SIGMA S5631
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
ReagentIsopropanolMerck MilliporeCatalog #109634
ReagentSodium acetateMerck MilliporeCatalog #1.06268.1000
Protocol materials
ReagentSodium acetateMerck Millipore (EMD Millipore)Catalog #1.06268.1000
ReagentEthanol
ReagentTris
ReagentSwinnex Filter HolderMerck MilliporeSigma (Sigma-Aldrich)Catalog #SX0004700
ReagentSilicon dioxide ~99% 0.5-10 µm (approx. 80% between 1-5 µm)Merck MilliporeSigma (Sigma-Aldrich)Catalog #SIGMA S5631
ReagentIsopropanolMerck Millipore (EMD Millipore)Catalog #109634
ReagentDurapore® Membrane Filter 5.0 µmMerck MilliporeSigma (Sigma-Aldrich)Catalog #SVLP04700
ReagentCentrifuge
ReagentTE bufferThermo Fisher Scientific
ReagentEDTA
ReagentMagnetic Funnel 300mL 47mmPallCatalog #4242
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
ReagentSodium ChlorideCatalog #PubChem CID: 5234
ReagentSodium acetateMerck Millipore (EMD Millipore)Catalog #1.06268.1000
ReagentIsopropanolMerck Millipore (EMD Millipore)Catalog #109634
ReagentSilicon dioxide ~99% 0.5-10 µm (approx. 80% between 1-5 µm)Merck MilliporeSigma (Sigma-Aldrich)Catalog #SIGMA S5631
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445
ReagentDurapore® Membrane Filter 5.0 µmMerck MilliporeSigma (Sigma-Aldrich)Catalog #SVLP04700
ReagentSwinnex Filter HolderMerck MilliporeSigma (Sigma-Aldrich)Catalog #SX0004700
ReagentMagnetic Funnel 300mL 47mmPallCatalog #4242
ReagentIsopropanolMerck Millipore (EMD Millipore)Catalog #109634
ReagentSodium acetateMerck Millipore (EMD Millipore)Catalog #1.06268.1000
ReagentSilicon dioxide ~99% 0.5-10 µm (approx. 80% between 1-5 µm)Merck MilliporeSigma (Sigma-Aldrich)Catalog #SIGMA S5631
Safety warnings
Wastewater is intrinsically hazardous, so we advise handling wastewater samples in a biosafety cabinet.
Before start
We developed this alternate procedure to allow the purification of wastewater RNA without access to a vacuum source or silica spin column. This companion method to "4S" enables highly efficient and extremely economical extraction of SARS-CoV-2 RNA from wastewater, but is more time and labor consuming. Using this procedure at the University of California Berkeley, we have captured and quantified SARS-CoV-2 and pepper mild mottle virus (PMMoV) present in a variety of San Francisco Bay Area raw wastewater influent samples and samples collected upstream of wastewater treatment plants. Results may vary depending on wastewater sample type and laboratory setting.

This procedure relies on centrifugation. In our laboratory setting, this procedure yields pure Wastewater RNA in approximately 6 hours.

In our laboratory, this purification method enables the detection of SARS-CoV-2 N and E gene RNA as well as PMMoV RNA via RT-qPCR probe-mediated detection. Depending on sample origin, we are able to recover an average of 25.7 ng RNA/mL of purified wastewater sample (min = 13.1 ng/mL, max = 58.2 ng/mL).
Preparing RNA wash buffers
Preparing RNA wash buffers
PrepareAmount1 L each of two wash buffers - Wash buffer #1 (4S-WB1) and #2 (4S-WB2), for later use during cleanup of RNA bound to silica particles.

Prepare a "Milk of Silica" suspension of dry silica.
4S-WB1 composition:
ReagentOriginal molarity/%Final molarity/%Volume per liter of buffer
NaCl5 M1.5 M300 mL
Ethanol100%20%200 mL
TRIS pH 7.21 M10 mM10 mL
Pure water (MilliQ or distilled)NANA490 mL
AddAmount490 mL water to sterile bottle
AddAmount300 mL ofConcentration5 Molarity (M) NaCl
AddAmount200 mL ofConcentration100 % volume Ethanol
AddAmount10 mL ofConcentration1 Molarity (M) Ph7.2 TRIS
Agitate to fully mix buffer solution
4S-WB2 composition:
ReagentOriginal molarity/%Final molarity/%Volume per liter of buffer
NaCl5 M100 mM20mL
Ethanol100%80%800mL
TRIS pH 7.21 M10 mM10mL
Pure water (MilliQ or distilled)NANA170mL
AddAmount170 mL water to sterile bottle
AddAmount20 mL ofConcentration5 Molarity (M) NaCl
AddAmount800 mL ofConcentration100 % volume Ethanol
AddAmount10 mL ofConcentration1 Molarity (M) Ph7.2 TRIS
Agitate to fully mix buffer solution
Prepare a "Milk of Silica" silica suspension by resuspending 5 grams of silicon dioxide powder in Amount5 mL of pure water. Scale "Milk of Silica" suspension volume by number of wastewater samples (5mL/sample).
ReagentSilicon dioxide ~99% 0.5-10 µm (approx. 80% between 1-5 µm)Merck MilliporeSigma (Sigma-Aldrich)Catalog #SIGMA S5631
"Milk of Silica" suspension (1g silicon dioxide/mL water)

Sample preparation, RNA preservation and particle lysis
Sample preparation, RNA preservation and particle lysis
Obtain aAmount40 mL wastewater sample in a sterile sample collection tube. Maintain atTemperature4 °C during transport to the lab.
Note
Sodium chloride and TE buffer (Go to step 4) can be added to sample immediately after collection. Our unpublished analysis demonstrates that Sodium chloride & TE buffer preserve RNA present in wastewater.


Spike a known volume and titer of bovine coronavirus (bCoV) into the wastewater sample as a recovery efficiency control. Agitate sample to fully mix bCoV with the wastewater sample. Agitate sample to fully mix bCoV or other spiked-in controls with the wastewater sample.
ReagentBovilis Coronavirus Calf VaccineMerck Animal HealthCatalog #16445

Note
Other recovery controls can be used instead of bCoV. Some candidates include Phi6 bacteriophage and coronavirus OC43. In addition, purified RNAs can be used to quantify the extraction efficiency of "free RNA".

AddAmount9.5 g of sodium chloride toAmount40 mL wastewater sample.
MakePh7.2 TE buffer (Concentration1 Molarity (M) TRIS,Concentration100 millimolar (mM) EDTA).
AddAmount400 µL of TE buffer to Amount40 mL wastewater sample.
Note
Here, NaCl lyses lipid-protein envelopes, denatures proteins and disrupts RNA-protein interactions. EDTA inhibits the enzymatic degradation of RNA by RNases present in wastewater and TRIS provides optimal buffering conditions for nucleic acids.


Agitate sample until all NaCl dissolves in the wastewater. Vortex or shake sample forDuration00:00:30 to promote lysis.

Raw wastewater containing NaCl, TRIS & EDTA.
(OPTIONAL) Heat inactivate sample atTemperature70 °C forDuration00:30:00 . Our unpublished analyses have shown that this step will not affect SARS-CoV-2 RNA enrichment and detection.
Filter the sample through a 5-um PVDF filter via syringe filtration or funnel top vacuum.

Syringe filter setup: Wastewater is filtered through a 47-mm reusable filter membrane holder.
ReagentDurapore® Membrane Filter 5.0 µmMerck MilliporeSigma (Sigma-Aldrich)Catalog #SVLP04700

ReagentSwinnex Filter HolderMerck MilliporeSigma (Sigma-Aldrich)Catalog #SX0004700

ReagentMagnetic Funnel 300mL 47mmPallCatalog #4242
Wastewater filtering through a 5-um PVDF filter in a Pall filter holder.
Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)
Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)
AliquotAmount40 mL filtrate into twoAmount20 mL aliquots. AddAmount20 mL ofConcentration70 % volume ethanol to eachAmount20 mL sample filtrate aliquot.

Filtered sample before ethanol addition. Filtrate should be semi-clear.
Agitate sample to mix ethanol and wastewater lysate.
Resuspend "Milk of Silica" suspension by inverting the slurry 10 times. AddAmount2.5 mL of the 1g/mL "Milk of Silica" slurry to each aliquot containingAmount40 mL of wastewater lysate with ethanol.




Invert tube with lysate & silica 10 times to mix. Incubate mixture at room temperature for Duration00:10:00 .
Note
In this step, the silica particles bind RNA present in the processed wastewater sample.


Centrifuge tubes containing silica & bound RNA at Centrifigation4000 x g, 4°C, 00:05:00 . The silica will form a firm pellet at the bottom of the tube. Remove the tubes from the centrifuge and decant and discard the supernatant.
Note
Here, the silica & bound RNA is precipitated to the bottom of the tube, separating it from the wastewater matrix.


AddAmount20 mL of 4S Wash buffer #1 (4S-WB1) to each silica pellet. Agitate or vortex tubes until silica is resuspended and appears milky.

Merge the two aliquot containingAmount20 mL 4S-WB1 and silica suspension by pouring the silica suspension from one tube into the other.

Centrifuge tubes containing silica, bound RNA and 4S-WB1 atCentrifigation4000 x g, 4°C, 00:05:00 . The silica will form a firm pellet at the bottom of the tube. Remove the tubes from the centrifuge and decant and discard the supernatant.

AddAmount40 mL of 4S Wash buffer #2 (4S-WB2) to the silica pellet. Agitate or vortex tubes until silica is resuspended and appears milky.
Centrifuge tubes containing silica, bound RNA and 4S-WB2Centrifigation4000 x g, 4°C, 00:05:00 . The silica will form a firm pellet at the bottom of the tube. Remove the tubes from the centrifuge and decant and discard the supernatant.

AddAmount40 mL of 4S Wash buffer #2 (4S-WB2) to the silica pellet. Agitate or vortex tubes until silica is resuspended and appears milky.
Centrifuge tubes containing silica, bound RNA and 4S-WB2Centrifigation4000 x g, 4°C, 00:05:00 . The silica will form a firm pellet at the bottom of the tube. Remove the tubes from the centrifuge and decant and discard the supernatant.
Vacuum aspirate any excess 4S-WB2 or allow tubes to incubate at room temperature forDuration00:10:00 to evaporate excess 4S-WB2.

Resuspend silica & RNA pellet inAmount20 mL of pure water (DNase and RNase-free) pre-warmed toTemperature37 °C . Vortex, agitate or pipette silica until fully resuspended. Allow silica & water suspension to incubate forDuration00:10:00 .
Note
Here, water elutes RNA from the silica particulate. The sample RNA is now present in the aqueous phase.

Centrifuge tubes containing silica & eluted RNA Centrifigation4000 x g, 37°C, 00:05:00 . The silica will form a firm pellet at the bottom of the tube and the RNA will be present in the aqueous phase. Pipette or decant the aqueous supernatant into a sterile conical bottom centrifugation-compatible (4000xg) tube for further concentration.
Note
This step separates the free, eluted RNA from the silica binding matrix, allowing downstream RNA concentration.

Concentration of eluted RNA (Isopropanol precipitation)
Concentration of eluted RNA (Isopropanol precipitation)
AddAmount20 mL ofConcentration100 % volume Isopropanol andAmount4 mL ofConcentration3 Molarity (M) Ph5.2 sodium acetate to the eluted RNA. Invert tube 10 times to mix solution and incubate mixture at room temperature forDuration00:10:00 .

ReagentIsopropanolMerck Millipore (EMD Millipore)Catalog #109634

ReagentSodium acetateMerck Millipore (EMD Millipore)Catalog #1.06268.1000

Note
Isopropanol and sodium acetate alongisde centrifugation precipitate the eluted RNA from the 20mL aqueous matrix.

Centrifuge sample atCentrifigation4000 x g, 4°C, 01:00:00 . A semi-translucent nucleic acid pellet will form at the bottom of the conical tube.
Note
Depending on sample type and source, the pellet may be brown or grey, as shown in the image in step 12.2

Carefully decant and discard the excess isopropanol & water from the nucleic acid pellet.

Side view of pellet after removal of isopropanol, water and sodium acetate mixture
Top view of pellet after removal of isopropanol, water and sodium acetate mixture
Wash pellet with ethanol by addingAmount40 mL Concentration75 % volume Ethanol to the nucleic acid pellet containing tube. Invert, vortex or agitate until the pellet loosens from the bottom of the tube and fully contacts the ethanol.
Note
Depending on sample type and origin, the pellet may fracture or remain intact during ethanol washing.

Re-precipitate nucleic acid pellet by centrifuging the sample atCentrifigation4000 x g, 4°C, 00:30:00 . After centrifugation, the nucleic acid pellet becomes visible at the bottom of the conical tube.
Carefully decant and discard as much supernatantConcentration75 % volume Ethanol as possible from the nucleic acid pellet. AddAmount1 mL ofConcentration70 % volume Ethanol to the nucleic acid pellet.

Using a pipette, resuspend the pellet in theAmount1 mL ofConcentration75 % volume Ethanol. Transfer the pellet and ethanol mixture to a 1.5mL microcentrifuge tube.
Note
To facilitate pellet transfer, use sterile scissors to cut the opening of 1mL pipette tips, allowing easier asipiration and transfer of the nucleic acid pellet.

After pellet transfer, centruge the microcentrifuge tube atCentrifigation5000 rpm, 4°C, 00:05:00 . The nucleic acid pellet will form at the bottom and side of the microcentrifuge tube.
Carefully pipette-aspirate the supernatantConcentration70 % volume Ethanol from the nucleic acid pellet.
Note
Use pipette tips with a small opening to remove excess ethanol without aspirating the pellet.

Open the lid of the microcentrifuge tube and incubate the tube atTemperature37 °C forDuration00:10:00 .
Note
This allows excess ethanol to evaporate, yielding ethanol-free RNA.

Resuspend the nucleic acid pellet inAmount200 µL of pure water or TE buffer. Vortex or pipette-mix the resuspended RNA to facilitate resuspension.
Note
It is possible for residual silica particles to remain in the final eluted RNA. In this case, briefly centrifuge the resuspended RNA and transfer the silica-free supernatant to a new sterile 1.5mL microfuge tube.


Storage
Storage
The eluted RNA is now ready for downstream analysis. Store RNA atTemperature4 °C for same-day use or freeze atTemperature-80 °C for later use and storage.