Apr 16, 2020
Sars-CoV2 RNA purification with homemade SPRI beads for RT-qPCR test
- Ayelet Rahat1,2,
- Miriam Adam3,2,
- Uri Shabi4,5,
- Moshe Cohen6,
- Daniel Kitsberg3,2,
- Malka Nissim-Rafinia1,
- Hagit Turm3,1,
- Agnes Klochendler7,
- Daphna Joseph-Strauss1,2,
- Israa Sharkia1,2,
- Matan Lotem1,2,
- Gavriel Fialkoff1,2,
- Ronen Sadeh1,2,
- Alon Chappleboim1,2,
- Yuval Dor7,
- Nir Friedman1,2,
- Dana Wolf7,
- Naomi Habib3,2
- 1Silberman Institute of Life Science, Hebrew University of Jerusalem;
- 2Rachel and Selim Benin School of Computer Science, Hebrew University of Jerusalem;
- 3Edmond and Lily Safra Center for Brain Sciences, Hebrew University of Jerusalem;
- 4Robiotec Ltd.;
- 5SciRobotics Ltd.;
- 6Neotec Bio;
- 7Institute for Medical Research Israel-Canada, The Hebrew University-Hadassah Medical School
- Coronavirus Method Development Community
Protocol Citation: Ayelet Rahat, Miriam Adam, Uri Shabi, Moshe Cohen, Daniel Kitsberg, Malka Nissim-Rafinia, Hagit Turm, Agnes Klochendler, Daphna Joseph-Strauss, Israa Sharkia, Matan Lotem, Gavriel Fialkoff, Ronen Sadeh, Alon Chappleboim, Yuval Dor, Nir Friedman, Dana Wolf, Naomi Habib 2020. Sars-CoV2 RNA purification with homemade SPRI beads for RT-qPCR test. protocols.io https://dx.doi.org/10.17504/protocols.io.beswjefe
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: April 07, 2020
Last Modified: April 16, 2020
Protocol Integer ID: 35382
Keywords: sars, covid19, spri, rna, extraction, purification, robot, automated, high throughput,
Abstract
The current SARS-CoV2 epidemic calls for large scale viral tests. The current testing procedure for the presence of virions involves several steps - pharyngeal/nasal swab, cell/viral lysis, RNA extraction, and an rtPCR assay targeting the viral genome and a human gene as an internal sample control. Improvement of any of these steps in terms of cost, processing time, or reagent availability, could result in a significant increase of testing capacity world-wide.
Here, we describe a rapid and efficient home made SPRI-based RNA extraction method from a lysed sample. Our approach can be fully automated, is quick (<30 min for 96 samples) and cheap (<1$ per sample), and was tested successfully on more than 250 clinical samples with approved rt-qPCR detection kits.
Guidelines
Note that different sources of viral samples arrive in different buffers.
This protocol was tested on samples derived by a swab into a standard Viral Transport Medium collection tube, and then mixed 1:1 with Zymo RNA/DNA shield for lysis.
This is a robot-compatible protocol. See attached zipped folder containing the robotic script and worktable for running the protocol on an EVO / Evoware platform and a pdf describing the robot configration and a human-readable protocol.
The protocol is designed to be adaptable to any robotic platform with a 96 channel arm and a manipulator arm capable of manipulating the plate containing the magnetic beads.
Materials
MATERIALS
NaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
PEG 8000 Powder (Polyethylene Glycol), 500gmPromegaCatalog #V3011
Trisodium citrate dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S1804
Water, nuclease free
1M Tris pH 7.5 Merck MilliporeSigma (Sigma-Aldrich)
Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophobic), 15 mLGE HealthcareCatalog #65152105050250
Tween 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1379
Ethanol
Lithium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #793620
HCl
Ethylenediaminetetraacetic acid (EDTA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #EDS
Lithium dodecyl sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #L4632
1M DL-Dithiothreitol solution (DTT)Merck MilliporeSigma (Sigma-Aldrich)Catalog #646563
1M Tris pH 8.0Merck MilliporeSigma (Sigma-Aldrich)Catalog #T2694
100mM Tris-HCl, pH 7.5
500mM LiCl
10mM EDTA
1% LiDS (also tested with 0.5% Triton x-100)
5mM DTT
Equipment needed:
Magnet for PCR strips or 96 well plate (DynaMag-96 Side Skirted Magnet #12027 from Invitrogen)
Equipment needed when processing by hand:
PCR strips or 96 well plate (minimum volume 150 µl)
Multichannel pipette (20ul and 200ul)
The robotic platform used for the protocol:
Tecan EVO150
MCA96 arm with disposable tips
RoMa with centric fingers
Inheco robotic shaker
Safety warnings
A risk assessment should be made when working with potenetially infectious specimen. Be sure that samples were properly handled and deactivated by certified personnal. Consult your local bio-safety staff.
Before start
- To prepare a large batch of home-made SPRI beads from the Sera-Mag™ Magnetic SpeedBeads follow our protocol - http://dx.doi.org/10.17504/protocols.io.bes2jege
- Take out SPRI beads from 4c storage for 30 minutes to arrive at room temperature
- Prepare fresh 80% EtOH (250 µl / sample)
- Verify you have enough Lysis/binding buffer (equal to the total volume of samples)
SPRI viral RNA Cleanup
SPRI viral RNA Cleanup
30m
30m
Sample dilution
Start with clinical samples collceted in virtal transport media and inactivated by dilution in lysis buffer. Add equal volume lysis/binding buffer to samples, e.g. for 28 µl, add 28 µl lysis/binding buffer
Note
The maximal volume in the process is x3.6 the sample volume, so use the maximum amount of sample that still allows efficient robotic pipettation, depending on your setup
1m
Bead Binding
- Add x0.8 volume SPRI beads to each diluted sample (e.g. 45 µl beads to 56 µl diluted sample)
- Mix by pipetting x10 times, avoid aeration during mixing to avoid foaming
- Incubate 00:10:00 at 25 °C , periodically mix by pippeting
- Magnetize until solution clears completely (~00:05:00 ) to make sure you do not loose beads
- Aspirate the supernatant slowly to avoid disturbing the magnetized beads and discard
Note
If beads can be seen in the tips after aspirating the supernatant or if beads are found in the waste consider the following reasons:
- Insufficient magnetization - Increase magnetization duration. The volume and buffer type affect the required duration.
- Bead scraping by the tip - Having the tip touch or scrape the beads during movement due to misalignment. A thinner tip can increase tolerance and help avoid this issue.
- Tip blockage and pressure buildup - If some of the tips press against the bottom of the well it will cause blockage and a buildup of negative pressure inside the tip. Once the tip moves up the pressure will cause a vortex inside the well which can disturb the beads. Have the tip at least 0.2mm above the bottom of the well and aspirate slowly
15m
80% EtOH Wash
- Add 120 µL of 80% EtOH
- Move the plate from one side of the magnet to the other to immerse the beads in the wash solution
- Wait for 00:00:30
- Remove the wash solution while the plate is on the magnet without disturbing the beads
- Repeat 80% EtOH wash (steps 3.1 - 3.4)
- Perform an extra aspiration of the residual EtOH with a fine tip
- Remove the plate from the magnet and air dry the beads to remove traces of EtOH (~00:02:30 minutes at 30 °C , until beads are dry by eye inspection). Avoid overdrying the beads.
10m
Elute
- Add 10mM Tris pH 8 (for 56ul beads 20 µL ) at 10 °C - 22 °C
- Resuspend the beads fully in the elution buffer by pipetting
- Incubate for 00:02:00 to allow full RNA elution
- Place the plate on the magnet and wait for the solution to clear (~00:01:00 )
- Keep on magnet, and transfer the supernatant to a new plate without disturbing the beads
Note
To resuspend the beads in the robotic system, we alternate between several steps:
- Wash the beads from the sides of the well - aspirate the elution liquid from the bottom of the well and dispense on the sides of the well above the z-line of the beads
- Shaking the plate
- Mixing by up-down pipetting about 0.5mm above the bottom of the well
4m