TITLE: RNA Extraction and Quality Assessment targeting SARS-CoV-2 from Wastewater Concentrates using Zymo Environ Water RNA Kit
AUTHORS: Tamara  Walsky, Padmini   Ramachandran, Amanda  Windsor, Maria  Hoffmann, Chris   Grim

[DESCRIPTION]
The Zymo Environ Water RNA kit has been tested and evaluated for the application of SARS-CoV-2 wastewater surveillance.  This kit was designed for water, wastewater, sludge, sewage, biofilm and other similar sample sources.  As such, minimal optimization was required for this application.  The kit incorporates an enrichment step that is accomplished with the use of Water Concentrating Buffer (WCB), which promotes the pelleting of viral capsids and protein-associated RNA, increasing SARS-CoV-2 RNA yields by more than 8-fold compared to without WCB. DNA/RNA Shield™ allows for safe handling as it has been validated for inactivation of pathogens including SARS-CoV-2. The kit yields highly concentrated, PCR inhibitor-free high quality RNA, applicable for many downstream applications including RT-qPCR, RT-ddPCR, and NGS.

[BEFORE_START]
Add 96 mL of 100% ethanol (or 104 mL of 95% ethanol) to the bottle labeled, RNA Wash Buffer, which contains 24 mL of RWB concentrate.

[STEPS]
SECTION: Sample Homogenization
1. 
 
Add 750 µl of DNA/RNA Shield™ to the concentrated wastewater sample (~200 µl, or to 250 µl liquid) sample to obtain a total of 1 ml of mixture. Mix well by pipetting up and down.


 

SECTION: Sample Homogenization
2. Add the 1 ml mixture to a ZR BashingBead™ Lysis Tube.
SECTION: Sample Homogenization
4. 12000 x g, 2 min, 25 °C Centrifuge the tube at 12,000 x g for 2 min 2 minutes to reduce foam.

SECTION: Sample Homogenization
5. Transfer 400 µl of the supernatant into an RNase-free tube. Proceed  to RNA Purification.
SECTION: RNA Purification
6. Add 1 volume of RNA Binding Buffer to the supernatant. Mix well.
 

SECTION: RNA Purification
8. Add 1 volume of ethanol (95-100%) to the flow-through in the Collection Tube from Step 2 and mix well by pipetting up and down.
SECTION: RNA Purification
9. Transfer the mixture into a new Zymo-Spin™ IIICG Column in a Collection Tube and centrifuge. If sample volume is greater than 800 µl, reload column. Discard the flow-through. Repeat again until all of the mixture is gone.
SECTION: RNA Purification
10. Add 400 µl of RNA Prep Buffer to the column and centrifuge. Then, transfer the column into an RNase-free microcentrifuge tube.
SECTION: RNA Purification
11. Add 100 µl of DNase/RNase-Free Water directly to the column matrix  and centrifuge.
SECTION: RNA Purification
12.  Place a Zymo-Spin™ III-HRC Filter into a new Collection Tube and add 600 µl of Prep Solution. Centrifuge at 8,000 x g for 3 minutes and discard the flow-through.
SECTION: RNA Purification
13. Transfer the eluted RNA from step 6 into a prepared Zymo-Spin™ III™ HRC Filter in a new RNase-free tube and centrifuge at exactly 16,000 x g for 3 minutes.
SECTION: RNA Purification
14. Add 200 µl of RNA Binding Buffer to the filtrate and mix well by  pipetting up and down.
SECTION: RNA Purification
15.  Add 300 µl of ethanol (95-100%) and mix well by pipetting up and  down.
SECTION: RNA Purification
16. Transfer the mixture into a Zymo-Spin IC Column in a new Collection Tube and centrifuge. Discard the flow-through.
SECTION: RNA Purification
17. Add 400 µl of RNA Prep Buffer to the column and centrifuge. Discard the flow-through.
SECTION: RNA Purification
20. Add 400 µl of RNA Wash Buffer to the column and centrifuge for 2  minutes to ensure complete removal of the wash buffer. Transfer the  column carefully into an RNase-free tube.
SECTION: RNA Purification
21. Add 50 µl of DNase/RNase-Free Water directly to the column matrix and centrifuge. The eluted RNA can be used immediately or stored at ≤ -70°C. If using immediately, keep RNA on ice at all times.
SECTION: Quality Assessment
22. Qubit 3.0 High Sensitivity RNA Quantification
Qubit Protocol: Document Connect (thermofisher.com)
SECTION: Quality Assessment
23. Femto Analyzer (or if using Fragment Analyzer, follow that protocol)
See Femto protocol here: Agilent FP-1201 Ultra Sensitivity RNA Quick Guide for Femto Pulse Systems

 

SECTION: Quality Assessment
24. Assessment of RNA quality using NanoDrop Microvolume Spectrophotometer
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24.2. Blank the NanoDrop with 1 µl of nuclease free water. Once blanked, wipe clean with a KimWipe.
SECTION: Quality Assessment
24.3. Pipette 1 µl of sample on to the NanoDrop. Measure and record 260/280 and 260/230. The closer each are to 2, the better the quality. Save the image if you want as well. Wipe the sample off the machine with a Kim Wipe before pipetting the next sample on.
SECTION: Quality Assessment
22.1. Take reagents out 30 minutes before use to reach room temperature.
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22.2. Calculate the total amount of buffer needed ((190 buffer*2 standards)+(199 buffer * x number of samples)= y ). Add an additional 10% to the volume calculated for y, to ensure there is enough buffer for all of your samples. Mix the appropriate amount of HS buffer with the concentrated dye (divide the total amount of buffer needed by 200, the result z is the amount of dye you need. Subtract y-z and that is the amount of buffer needed, pipette z amount of dye in to the buffer. Vortex.
SECTION: Quality Assessment
22.3. Pipette the correct amount of buffer to a corresponding Qubit tube. (190 μL for standards, 199 μL for samples.) Pipette 10 μL of Standards 1 and 2 in to their respective tubes and 1 μL of sample in to the respective tubes. Vortex all the tubes.
SECTION: Quality Assessment
22.5. Place standards in to the Qubit reader, moving on to your samples after. Record results.
SECTION: Quality Assessment
23.1. If this is a new kit, set up the ladder as described in the protocol: 

a. Thaw the RNA ladder on ice, agitate solution to ensure it is properly mixed and centrifuge vial prior to dispensing. Using the provided Eppendorf LoBind 0.5 mL tubes, aliquot 3μL of the RNA ladder per tube into 5 tubes and store the aliquots at -80°C.
b. Thaw an RNA ladder aliquot on ice.
c. Heat-denature the entire 3 μL aliquot of the RNA ladder at 70°C for 2 min, immediately cool to 4°C and keep on ice.
d. Transfer 2 μL of denatured RNA ladder to a fresh Lo-Bind tube and add 18 μL of the provided FP RNA Dilution Buffer (FP-6501-0003); mix thoroughly. This is now the working RNA ladder solution.
e. Store any unused portion of the working RNA ladder at -80°C. The working RNA ladder should not need to be heat denatured again. Each diluted aliquot is good for 5 freeze/thaw cycles.

If ladder is diluted, take out of the freezer and keep on ice until use.

SECTION: Sample Homogenization
3. Secure in a vortex genie fitted with a 2 ml tube holder assembly and process for 5 minutes at level 4.

SECTION: RNA Purification
7. Transfer the mixture into a Zymo-Spin™ IIICG Column in a Collection Tube and centrifuge. If sample volume is greater than 800 µl, reload column.  Save the flow-through!
SECTION: Quality Assessment
23.2. Calculate the dilution needed for the samples based on the Qubit result. The Femto takes 15 pg/µL - 250 pg/µL input RNA. Make appropriate dilutions. DO NOT heat-denature sample RNA as described in the protocol.

Dilution set up:
(x ng/µL * 1000)= y pg/µL
y pg/µL* dilution factor (either 0.1,0.01,0.001)= final concentration in pg/µL, should be between 15 pg/µL - 250 pg/µL
Then take 1 µL of sample in to 9/99/999 µL of NF water. Mix by flicking and spin down.
SECTION: Quality Assessment
23.3. Using a clean RNase-free 96-well sample plate, pipette 18 μL of FP US RNA Diluent Marker (DM) solution (FP-8201)  to each well in a row that is to contain sample or RNA ladder.  Fill any unused wells within the row of the sample plate with 20 μL of BF-P25 Blank Solution.
SECTION: Quality Assessment
23.4. RNA ladder: The RNA ladder must be run in parallel with the samples for each experiment to ensure accurate  quantification. a) Pipette 2 μL of working RNA ladder Solution (prepared above) into the 18 μL of DM solution in the designated  ladder well (Well #12) of each row to be analyzed. b) Mix the contents of the well using the pipette by aspiration/expulsion in the pipette tip
SECTION: Quality Assessment
23.5. Samples: Pipette 2 μL of each DNA sample into the 18 μL of DM Solution in the respective wells of the Sample Plate;  mix the contents of the well using the pipette by aspiration/expulsion in the pipette tip.
SECTION: Quality Assessment
23.6. After mixing sample/RNA ladder and DM solution in the sample plate, centrifuge the plate to remove any air bubbles.  Check the wells of the sample plate to ensure there are no air bubbles trapped in the bottom of the wells. The  presence of trapped air bubbles can lead to injection failures.
SECTION: Quality Assessment
23.7.  For best results, run the plate as soon as possible. If the sample plate will not be used immediately, cover the sample  plate with a RNase-free plate seal, store at 2-8°C and use within the same day. Bubbles may develop in the sample  wells while sitting at 2-8°C, make sure to centrifuge the plate again and remove the seal before placing the plate into  the instrument. The sample plate should be analyzed within a day after preparation. To run the samples, place the plate in one of the three sample plate trays (Drawers 4-6 from the top) of the Femto Pulse System. Run using the total RNA method.
SECTION: RNA Purification
18. 
 

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19. Add 700 µl of RNA Wash Buffer to the column and centrifuge. Discard the flow-through.
SECTION: Quality Assessment
22.4. Incubate all samples for at least 2 minutes.


 

SECTION: Quality Assessment
24.1. Select RNA as the sample type.