Apr 27, 2020
Version 2
Manual Silane magnetic bead-based high throughput protocol for SARS-CoV-2 RNA extraction V.2
- Ainhoa Goñi-Salaverri1,
- Jose A. Rodriguez1,
- Nicholas Weber2,
- Juan Pablo Unfried1,
- Josepmaria Argemi3,
- 4,
- David Lara Astiaso1,
- 5
- 1Cima Universidad de Navarra, Pamplona, Spain;
- 2Vivet Therapeutics SL, Pamplona, Spain;
- 3Liver Unit. Internal Medicine Department. Clinica Universidad de Navarra, Pamplona, Spain;
- 4Division of Medicine - Gastroenterology and Hepatology Department, University of Pittsburgh, PA, USA;
- 5Cambridge Stem Cell Institute, Cambridge, England
- Coronavirus Method Development Community
Protocol Citation: Ainhoa Goñi-Salaverri, Jose A. Rodriguez, Nicholas Weber, Juan Pablo Unfried, Josepmaria Argemi, , David Lara Astiaso, 2020. Manual Silane magnetic bead-based high throughput protocol for SARS-CoV-2 RNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bfmajk2e
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 27, 2020
Last Modified: April 27, 2020
Protocol Integer ID: 36226
Keywords: SARS-CoV2, RNA, extraction, purification, high throughput, Silane ferrimagnetic beads, COVID-19, current testing for severe acute respiratory syndrome coronavirus, rna extraction, testing tools for urgent disease diagnosis, purification of the viral genetic material, coronavirus, identification of viral genetic material, severe acute respiratory syndrome coronavirus, viral genetic material, manual silane magnetic bead, patient sampling to diagnosis, silane ferrimagnetic bead, molecular diagnostic, high throughput protocol for sar, protocol for sar, ferrimagnetic bead, rna, patient sampling, magnetic bead
Abstract
The COVID-19 pandemic has presented an unprecedent challenge to develop and validate testing tools for urgent disease diagnosis. Current testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via molecular diagnostics is woefully inadequate due to myriad issues with cross-entity coordination, reagent availability, and labor- and time-intensive protocols. The bottleneck in the workflow from patient sampling to diagnosis is not in the identification of viral genetic material (RT-qPCR) but in the collection, processing, and purification of the viral genetic material (patient sampling and RNA extraction). The majority of tests performed in laboratories lacking sophisticated automation require individual sample processing that severely limits the possibilities of scale-up. Thus, throughput depends on the availability of trained operator person-hours.
We have developed a protocol for SARS-CoV-2 RNA extraction based on silane ferrimagnetic beads that is easily scaled up to multiwell culture plates with the processing done using multichannel pipettes. This protocol significantly reduces the hands-on time per sample and increases throughput such that an individual operator can process 100-200 samples per hour.
Attachments
Materials
MATERIALS
Buffer RLT PlusQiagenCatalog #1053393
80% Ethanol
DynaMag™-96 side skirted magnet Life TechnologiesCatalog #12027
100% Ethanol
DynaMag™-2 MagnetThermo FisherCatalog #12321D
Graduated Safelock Microcentrifuge Tubes, 2.0mLThermo FisherCatalog #3458
Dynabeads™ MyOne™ SilaneThermo FisherCatalog #37002D
10 mM Tris pH7.5
Swabs
96-Well Semi-Skirted PlatesThermofisherCatalog #AB0900
Polystyrene Solution ReservoirsLabdexCatalog #03-103PSRL
Pippette Tips GP LTS 1000 ul (filter)Mettler ToledoCatalog #30389272
Pippette Tips GP LTS 200 ul (filter)Mettler ToledoCatalog #30389276
Pippette Tips GP LTS 20 ul (filter)Mettler ToledoCatalog #30389274
Pippette Tips GP LTS 20 ulMettler ToledoCatalog #30389270
LTS Pipette L-1000XLS Mettler ToledoCatalog #17014382
Multi Pipette L8-200XLS Mettler ToledoCatalog #17013805
Multi Pipette L8-20XLS Mettler ToledoCatalog #17013803
Vacuum station
Biosafety Level 2 Laminar Flow Hood
Troubleshooting
Safety warnings
Biosafety
- Be sure to wear the appropriate equipment: lab coat, two pairs of gloves, face mask and glasses.
- Use UV light to inactivate any remaining viral particles inside the laminar flow hood.
Before start
Reagent preparation and considerations
SAMPLE COLLECTION
- Aliquot 800 l of RLT plus buffer into 2 mL safe-lock ED tubes
- Use safe-lock tubes for sample collection and transport to prevent sample leakage
RNA EXTRACTION
- Pre-heat an aliquot (~10 mL) of 10 mM Tris-Cl pH 7.5 to 37C.
- Prepare fresh 80% ethanol solution
- Use multichannel pipettes to increase the throughput (we use Rainin pipettes)
- Use filter tips for the entire process except for the aspiration steps
- If possible, install a vacuum station with an 8-channel adaptor
Sample collection
Following oropharyngeal and/or nasopharyngeal swab collection, place the swab directly into a 2 mL safe-lock tube containing 800 l of RLT-plus buffer (Qiagen).
* When collected in RLT plus the viral particles become inactive. This provides further biosafety for the process. However, samples should be always considered as extremely biohazardous and handled with extreme caution inside a Level 2 biosafety hood.
Cut the swab’s stem and close the tube.
If possible, store samples at 4C or -20C.
* Though, we have seen that when placed in RLT plus buffer the RNA integrity of the samples is preserved at room temperature for 24-48 hours.
Sample transfer into deep 96-well plates
20m
Let samples thaw for 20 minutes at Room Temperature (if transported or stored frozen).
* All the process should be performed inside a Biosafety Level 2 Cabinet following the WHO guidelines for processing SARS-CoV-2 samples.
* Use Eppendorf deep-well plates with capacity for 250 l.
Open safe-lock tube and transfer 150 μl of sample in RLT to a well in the 96-well plate.
* When processing 48 or fewer samples assemble the samples in alternating columns (leaving an empty column between sample columns). This minimizes the risk of contamination and facilitates the process.
Return the tube with the remaining sample to the original box and carefully record the position of each sample in the 96-well plate.
* This process is best performed by teams of two with one person transferring the samples and the second recording the positions.
Silane Bead preparation (performed during swab sample thawing)
2m
Silane beads (stored at 4C) are washed and resuspended according to the following method (performed outside the flow hood):
Aliquot 15 μl of beads per sample into a 2 mL ED tube (calculate a 15% overage, e.g. 15 extra reactions per each 96 samples: 111x15 l = 1,665 l of Silane beads).
Concentrate beads to the tube wall using a DynaMag-2 magnet; remove bead storage suspension media.
Wash 1X with 1 mL of RLT-plus buffer, and remove as in the previous step.
Resuspend in 10 μl of RLT-plus buffer per sample.
Pipette the required amount of pre-washed Silane beads (10 μl/sample) into a 15 mL tube (calculate a 15% overage, e.g. 15 extra reactions per each 96 samples: 111x10 μl = 1,110 μl of pre-washed Silane beads).
Add 70 μl 100% EtOH per sample (7,770 μl for 96 samples), vortex and keep at room temperature:
| Reagent | x1 sample | x96 samples (+15 of excess) | |
| Pre-washed Silane beads | 10 μl | 1,110 μl | |
| 100% EtOH | 70 μl | 7,770 μl | |
| Total | 80 μl | 8,880 μl |
Silane bead-mediated RNA purification
30m
Vortex the Silane bead-ethanol mixture and pour it into a reservoir.
Use a multichannel pipette to add 80 l of the Silane bead/ethanol into each sample well and mix well by pipetting up and down 15 times.
Incubate for 5 minutes at room temperature.
Move the plate onto a DynaMag-96 side skirted magnet and incubate for 3 minutes at room temperature.
Once beads are strongly magnetized and with the plate still on the magnet, aspirate the supernatant with an 8-channel vacuum aspirator.
* Alternatively use a multichannel pipette.
With the plate on the magnet, use the multichannel pipette to add 200 μl of 80% EtOH from another reservoir and incubate for 30 seconds.
* Do not re-suspend the beads after adding the ethanol.
Aspirate the ethanol with an 8-channel vacuum aspirator .
* Alternatively use a multichannel pipette.
Repeat the addition of 150 μl of 80% EtOH and incubate for 30 seconds.
* Do not re-suspend the beads after adding the ethanol.
Slowly aspirate the ethanol with an 8-channel vacuum aspirator.
*It is very important to avoid leaving small droplets of ethanol 80% that may inhibit the subsequent RT-qPCR step.
Take off the plate from the magnet and let the beads dry inside the hood for 1 minute.
* Avoid over drying the beads of they will become sticky and difficult to re-suspend.
Add 30-50 l of preheated 10 mM Tris pH 7.5 (from a reservoir) and resuspend the beads by pipetting up and down thoroughly.
* If samples become difficult to resuspend, incubate for 3 minutes at 37C and then try to resuspend the beads.
Incubate at 37C for 3 minutes to increase the yield of the elution.
* This step may be skipped if yield is sufficient without it.
Magnetize the plate again by placing it onto the DynaMag-96 side skirted magnet for 1 minute.
Transfer supernatant into a new 96-well plate.
Eluted RNA is ready for the RT-qPCR reaction.