TITLE: Direct wastewater RNA extraction via the "Milk of Silica (MoS)" method - A companion method to "Sewage, Salt, Silica and SARS-CoV-2 (4S)"
AUTHORS: Oscar 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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The following protocol describes the "4S" (</span><a href="http://dx.doi.org/10.17504/protocols.io.biwekfbe" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Sewage, Salt, Silica and SARS-CoV-2</span></a><span>) 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.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/ffce19a396882be605eb2c93185943f2745a7f7440126d7b6b804da64064cc05/cfpibdz6x.png" /></div></div>

[STEPS]
?. [Preparing RNA wash buffers]
Prepareeach 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.
1 L
?. [Preparing RNA wash buffers]
4S-WB1 composition:   ABCD1ReagentOriginal molarity/%Final molarity/%Volume per liter of buffer2NaCl5 M1.5 M300 mL3Ethanol100%20%200 mL4TRIS pH 7.21 M10 mM10 mL5Pure water (MilliQ or distilled)NANA490 mLAddwater to sterile bottleAddofNaClAddof EthanolAddofTRISAgitate to fully mix buffer solution
ABCD1ReagentOriginal molarity/%Final molarity/%Volume per liter of buffer2NaCl5 M1.5 M300 mL3Ethanol100%20%200 mL4TRIS pH 7.21 M10 mM10 mL5Pure water (MilliQ or distilled)NANA490 mL
490 mL
300 mL
200 mL
10 mL
?. [Preparing RNA wash buffers]
4S-WB2 composition:    ABCD1ReagentOriginal molarity/%Final molarity/%Volume per liter of buffer2NaCl5 M100 mM20mL3Ethanol100%80%800mL4TRIS pH 7.21 M10 mM10mL5Pure water (MilliQ or distilled)NANA170mLAddwater to sterile bottleAddofNaClAddof EthanolAddofTRISAgitate to fully mix buffer solution
ABCD1ReagentOriginal molarity/%Final molarity/%Volume per liter of buffer2NaCl5 M100 mM20mL3Ethanol100%80%800mL4TRIS pH 7.21 M10 mM10mL5Pure water (MilliQ or distilled)NANA170mL
170 mL
20 mL
800 mL
10 mL
?. [Sample preparation, RNA preservation and particle lysis]
Obtain awastewater sample in a sterile sample collection tube. Maintain atduring transport to the lab.
40 mL
4 °C
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.
?. [Sample preparation, RNA preservation and particle lysis]
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.
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".
?. [Sample preparation, RNA preservation and particle lysis]
Addof sodium chloride towastewater sample. MakeTE buffer (TRIS,EDTA). Addof TE buffer to wastewater sample.
9.5 g
40 mL
400 µl
40 mL
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.
?. [Sample preparation, RNA preservation and particle lysis]
(OPTIONAL) Heat inactivate sample atfor. Our unpublished analyses have shown that this step will not affect SARS-CoV-2 RNA enrichment and detection.
70 °C
?. [Sample preparation, RNA preservation and particle lysis]
Filter the sample through a 5-um PVDF filter via syringe filtration or funnel top vacuum.
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Aliquotfiltrate into twoaliquots. Addofethanol to eachsample filtrate aliquot.
40 mL
20 mL
20 mL
20 mL
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Agitate sample to mix ethanol and wastewater lysate.
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Addof 4S Wash buffer #2 (4S-WB2) to the silica pellet. Agitate or vortex tubes until silica is resuspended and appears milky.
40 mL
?. [Concentration of eluted RNA (Isopropanol precipitation)]
AddofIsopropanol andofsodium acetate to the eluted RNA. Invert tube 10 times to mix solution and incubate mixture at room temperature for.
20 mL
4 mL
Isopropanol and sodium acetate alongisde centrifugation precipitate the eluted RNA from the 20mL aqueous matrix.
?. [Concentration of eluted RNA (Isopropanol precipitation)]
Wash pellet with ethanol by addingEthanol 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.
40 mL
Depending on sample type and origin, the pellet may fracture or remain intact during ethanol washing.
?. [Concentration of eluted RNA (Isopropanol precipitation)]
After pellet transfer, centruge the microcentrifuge tube at. The nucleic acid pellet will form at the bottom and side of the microcentrifuge tube.
Centrifuge: 5000 33, 5 min, 4 10
?. [Concentration of eluted RNA (Isopropanol precipitation)]
Carefully pipette-aspirate the supernatantEthanol from the nucleic acid pellet.
Use pipette tips with a small opening to remove excess ethanol without aspirating the pellet.
?. [Concentration of eluted RNA (Isopropanol precipitation)]
Open the lid of the microcentrifuge tube and incubate the tube atfor.
37 °C
This allows excess ethanol to evaporate, yielding ethanol-free RNA.
?. [Preparing RNA wash buffers]
Prepare a "Milk of Silica" silica suspension by resuspending 5 grams of silicon dioxide powder in of pure water. Scale "Milk of Silica" suspension volume by number of wastewater samples (5mL/sample).
5 mL
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Resuspend "Milk of Silica" suspension by inverting the slurry 10 times. Addof the 1g/mL "Milk of Silica" slurry to each aliquot containingof wastewater lysate with ethanol.
2.5 mL
40 mL
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Invert tube with lysate & silica 10 times to mix. Incubate mixture at room temperature for .
In this step, the silica particles bind RNA present in the processed wastewater sample.
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Centrifuge tubes containing silica, bound RNA and 4S-WB1 at. 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.
Centrifuge: 4000 34, 5 min, 4 10
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Merge the two aliquot containing4S-WB1 and silica suspension by pouring the silica suspension from one tube into the other.
20 mL
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Addof 4S Wash buffer #1 (4S-WB1) to each silica pellet. Agitate or vortex tubes until silica is resuspended and appears milky.
20 mL
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Centrifuge tubes containing silica & bound RNA at . 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.
Centrifuge: 4000 34, 5 min, 4 10
Here, the silica & bound RNA is precipitated to the bottom of the tube, separating it from the wastewater matrix.
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Remove tubes from centrifuge and decant supernatant from the silica pellet.
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Centrifuge tubes containing silica, bound RNA and 4S-WB2. 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.
Centrifuge: 4000 34, 5 min, 4 10
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Resuspend silica & RNA pellet inof pure water (DNase and RNase-free) pre-warmed to. Vortex, agitate or pipette silica until fully resuspended. Allow silica & water suspension to incubate for.
20 mL
37 °C
Here, water elutes RNA from the silica particulate. The sample RNA is now present in the aqueous phase.
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Vacuum aspirate any excess 4S-WB2 or allow tubes to incubate at room temperature forto evaporate excess 4S-WB2.
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Centrifuge tubes containing silica & eluted RNA . 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.
Centrifuge: 4000 34, 5 min, 37 10
This step separates the free, eluted RNA from the silica binding matrix, allowing downstream RNA concentration.
?. [Concentration of eluted RNA (Isopropanol precipitation)]
Centrifuge sample at. A semi-translucent nucleic acid pellet will form at the bottom of the conical tube.
Centrifuge: 4000 34, 60 min, 4 10
Depending on sample type and source, the pellet may be brown or grey, as shown in the image in step 12.2
?. [Concentration of eluted RNA (Isopropanol precipitation)]
Carefully decant and discard the excess isopropanol & water from the nucleic acid pellet.
?. [Concentration of eluted RNA (Isopropanol precipitation)]
Re-precipitate nucleic acid pellet by centrifuging the sample at. After centrifugation, the nucleic acid pellet becomes visible at the bottom of the conical tube.
Centrifuge: 4000 34, 30 min, 4 10
?. [Concentration of eluted RNA (Isopropanol precipitation)]
Carefully decant and discard as much supernatantEthanol as possible from the nucleic acid pellet. AddofEthanol to the nucleic acid pellet.
1 mL
?. [Concentration of eluted RNA (Isopropanol precipitation)]
Using a pipette, resuspend the pellet in theofEthanol. Transfer the pellet and ethanol mixture to a 1.5mL microcentrifuge tube.
1 mL
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.
?. [Concentration of eluted RNA (Isopropanol precipitation)]
Resuspend the nucleic acid pellet inof pure water or TE buffer. Vortex or pipette-mix the resuspended RNA to facilitate resuspension.
200 µl
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.
?. [Sample preparation, RNA preservation and particle lysis]
Agitate sample until all NaCl dissolves in the wastewater. Vortex or shake sample forto promote lysis.
?. [Storage]
The eluted RNA is now ready for downstream analysis. Store RNA atfor same-day use or freeze atfor later use and storage.
4 °C
-80 °C
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Addof 4S Wash buffer #2 (4S-WB2) to the silica pellet. Agitate or vortex tubes until silica is resuspended and appears milky.
40 mL
?. [Direct RNA extraction via addition of silica slurry (RNA Binding, Washing, Eluting)]
Centrifuge tubes containing silica, bound RNA and 4S-WB2. 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.
Centrifuge: 4000 34, 5 min, 4 10