Mar 11, 2023

Public workspaceSARS-CoV-2 RNA extraction with Ceres Nanotrap and Zymo Environ Water 

DOI
https://dx.doi.org/10.17504/protocols.io.14egn26qqg5d/v1
SARS-CoV-2 RNA extraction with Ceres Nanotrap and Zymo Environ Water
  • Amanda Windsor1,
  • kathryn.judy 1,
  • Tamara Walsky1,
  • Padmini Ramachandran1,
  • Chris Grim1,
  • Maria Hoffmann1
  • 1FDA CFSAN
Amanda Windsor
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn26qqg5d/v1
Protocol Citation: Amanda Windsor, kathryn.judy , Tamara Walsky, Padmini Ramachandran, Chris Grim, Maria Hoffmann 2023. SARS-CoV-2 RNA extraction with Ceres Nanotrap and Zymo Environ Water . protocols.io https://dx.doi.org/10.17504/protocols.io.14egn26qqg5d/v1
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: In development
We are still developing and optimizing this protocol
Created: December 07, 2022
Last Modified: March 11, 2023
Protocol Integer ID: 73627
Keywords: SARS-CoV-2, Ceres Nanotrap, Zymo Environ Water, RNA, wastewater, rna extraction, ceres nanotrap, based virus capture, virus capture, zymo environ water, zymo dnase step, wastewater, extraction, dnase, rna, virus
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Abstract
This protocol uses the Ceres Nanotrap® particle-based virus capture and concentration method for 10mL of wastewater followed by extraction with the Zymo Environ™ Water RNA extraction kit with a Zymo DNase step.
Guidelines
When developing this protocol, we extracted at least 2 replicates of each wastewater sample to ensure we had ample RNA for downstream processes

RNA extraction is performed at room temperature and centrifugation at 10,000-16,000 x g for 30s
Materials
  1. Wastewater sample
Reagents and Kits
  1. Ceres Nanosciences Nanotrap® Magnetic Viral Particles (Ceres Nanosciences: SKU 44202)
  2. Ceres Nanosciences Nanotrap® Enhancement Reagent 2 (Ceres Nanosciences: SKU 10112)
  3. Magnetic separator for 15 mL conical tubes, such as Invitrogen™ DynaMag™-2 Magnet (ThermoFisher Cat# 12-301-D)
  4. Magnetic separator for 2mL micro centrifuge tubes, such as Invitrogen™ DynaMag™-2 Magnet (ThermoFisher Cat# 12-321-D)
  5. Zymo Environ™ Water RNA Kit (Zymo Research: R2042)
  6. Zymo DNA/RNA Shield™ (Zymo Research: R1100-50 or R1100-250)
  7. Zymo DNase Set 1 (Zymo Research: E1010)
Equipment
  1. Programable Heat Block
  2. Mini vortex mixer
  3. Mini Centrifuge (Max capable of 16,000 x g & fits 1.5/2mL tubes)
  4. tube rotator (e.g. Fisherbrand Mini Tube Rotator Cat 88-861-05 or similar)
Consumables
  1. 100% absolute ethanol
  2. DNase/RNase Free Water
  3. 15mL conical tubes
  4. 1.5 or 2mL microcentrifuge tubes
  5. 100-1000uL pipette
  6. 20-200uL pipette
  7. 100-1000uL filtered pipette tips
  8. 20-200uL filtered pipette tips
  9. serological pipetting aid
  10. 10mL serological pipettes
  11. 5mL serological pipettes
  12. 1mL serological pipettes

Troubleshooting
Safety warnings
Please refer to Safety Data Sheets (SDS) for health and environmental hazards.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
Before start
  • Store the reagents separately from RNA/TNA (total nucleic acid) samples.
  • Use a clean designated work area and separate pipettes for pre- and post-extraction steps to minimize the potential for cross-contamination
  • Wear a lab coat and protective eyewear.
  • Wear gloves and change them often.
  • Prevent contamination by using aerosol-resistant pipette tips.
Before you start
Turn on heat blockTemperature95 °C
Ensure appropriate volume of DNase I is available (5µL per sample), or make new aliquots
Add Amount275 µL DNase/RNase-Free water to reconstitute lyophilized DNase I (1U/µL)

Note
Aliquot reconstituted DNase I in volumes appropriate for your lab's throughput (e.g., 15-20µL aliquots in 0.5mL microcentrifuge tubes) to avoid multiple freeze/thaw cycles

Viral Capture with Nanotrap® Particles
30m
Shake wastewater bottle to mix then let sit Duration00:00:45

45s
Using a 10mL serological pipette, carefully pipette Amount10 mL of wastewater into a 15mL conical tube

Note
Input volumes of 20, 30, and 40mL have also been tested. See note in step 7 for volume of nanotrap particles to add based on starting volume

Add Amount100 µL of Nanotrap® Enhancement Reagent 2 (ER2) and invert 15mL tube 2-3 times to mix

Re-suspend Nanotrap® particles by inverting the bottle 5 times

Add Amount150 µL Nanotrap® particles to the sample

Note
the volume of nanoparticles for different starting volumes of wastewater are as follows:

Input Wastewater (mL)Nanotrap Particles (µL)
20300
30450
40600
volume of nanoparticles for different starting volumes of wastewater


Incubate samples TemperatureRoom temperature Duration00:10:00 with constant rotation

10m
Place samples on magnetic rack to separate Magnetic Nanotrap® particles from the sample - at least Duration00:02:00

2m
After beads have settled, use a 5mL serological pipette to remove all of the supernatant without disturbing the pelleted beads


Add Amount1 mL of DNAse/RNAse Free water to the tube

Remove tube from magnet and re-suspend the pelleted beads using a 100-1000uL pipette
Transfer suspended beads to a 1.5mL microcentrifuge tube
Place microcentrifuge tube on the 2mL tube-compatible magnetic rack
Incubate until the beads have settled - at least Duration00:02:00

2m
Remove supernatant with a 100-1000uL pipette without disturbing the pellet. Remove any small amount of remaining supernatant with a smaller pipette tip (e.g. 2-20uL pipette)


Remove the tubes from the magnet and re-suspend the pellet with Amount375 µL Zymo DNA/RNA Shield and Amount125 µL Zymo DNase/RNase-Free water from the Zymo Environ Water RNA Kit
Incubate the samples at Temperature95 °C for Duration00:05:00

5m
While samples are incubating, add Amount400 µL Zymo RNA Binding Buffer from the Zymo Environ Water RNA Kit to new 1.5mL tubes (one per sample)

Remove tubes from heat block and place on a magnetic rack and allow beads to settle until supernatant is clear - at least Duration00:02:00


Note
Collect any liquid from caps by brief centrifugation prior to placing the tubes on the magnetic rack

2m
Reset heat block temperature to Temperature27 °C

Transfer Amount400 µL of supernatant to the corresponding tube prepared in step 15.1 and mix by gentle pipetting
Note
sample tubes volume = 800µL


Zymo Environ™ Water RNA Kit
5m
Transfer entire sample to a Zymo-Spin™ IIICG Column in a clean collection tube and centrifuge Centrifigation10000 x g, Room temperature, 00:00:30 and keep the flow-through

Note
Label both the spin column and collection tube

30s
Add 800uL of ethanol (95-100%) to the flow-through in the collection tube from step 18 and mix well by gentle pipetting
Note
sample volume = 1600µL

Transfer Amount800 µL into a new Zymo-Spin™ IIICG Column in a clean collection tube and centrifuge Centrifigation10000 x g, Room temperature, 00:00:30 and discard the flow through

30s
Repeat step 19 with the remaining Amount800 µL of sample using the same collection tube

Add Amount400 µL of RNA Prep Buffer to the column and centrifuge Centrifigation10000 x g, Room temperature, 00:00:30 and discard the flow-through
30s
Transfer column to an RNase-Free 1.5mL microcentrifuge tube
Add Amount100 µL Zymo DNase/RNase-Free water directly to the column matrix and centrifuge Centrifigation10000 x g, Room temperature, 00:00:30 and keep the flow-through for step 24

30s
Place a Zymo-Spin III-HRC Filter into a new collection tube and add Amount600 µL Prep Solution Centrifuge Centrifigation8000 x g, Room temperature, 00:03:00 discard the flow-through

3m
transfer the column to an RNase-Free 1.5mL microcentrifuge tube
Transfer the eluted RNA from step 22 into the Zymo-Spin III-HRC filter prepared in step 23 and Centrifigation16000 x g, Room temperature, 00:03:00 keep the flow-through

3m
Add Amount200 µL RNA Binding Buffer to the filtrate and mix well by gently pipetting up and down.
Add Amount300 µL of ethanol (95-100%) to the filtrate + RNA Binding Buffer and mix well by gently pipetting up and down.
Transfer the mixture into a Zymo-Spin IC column in a collection tube and Centrifigation10000 x g, Room temperature, 00:00:30 discard the flow-through

30s
Add Amount400 µL RNA Wash Buffer to column and Centrifigation10000 x g, Room temperature, 00:00:30 Discard flow-through

30s
Add Amount5 µL DNase I and Amount35 µL DNA Digestion Buffer to the column matrix

Incubate Temperature27 °C Duration00:20:00

20m
Add Amount400 µL of RNA Prep Buffer to the column and Centrifigation10000 x g, Room temperature, 00:00:30 discard the flow-through.
30s
Add Amount700 µL of RNA Wash Buffer to the column and Centrifigation10000 x g, Room temperature, 00:00:30 discard the flow-through.

30s
Add Amount400 µL of RNA Wash Buffer to the column and Centrifigation10000 x g, Room temperature, 00:02:00 to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube.
2m
Add Amount50 µL of DNase/RNase-Free Water directly to the column matrix
and Centrifigation10000 x g, Room temperature, 00:00:30 The eluted RNA can be used immediately or stored at Temperature-70 °C .

30s