TITLE: Manual Silane magnetic bead-based high throughput protocol for SARS-CoV-2 RNA extraction
AUTHORS: Ainhoa Goñi-Salaverri, Jose A. Rodriguez, Nicholas Weber, Juan Pablo Unfried, Josepmaria Argemi,  , David Lara Astiaso,  

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. </div><div class = "text-block">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.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/d8304a24a21b08f45f0dba1f25f71d49f2590013af069177e904a82e46875a82/b63jbcw2p.png" /></div></div>

[STEPS]
?. [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.
?. [Sample collection]
Cut the swab’s stem and close the tube.
?. [Sample collection]
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]
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.
?. [Sample transfer into deep 96-well plates]
Open safe-lock tube and transfer 150 μl of sample in RLT to a well in the 96-well plate.
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							* 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.
?. [Sample transfer into deep 96-well plates]
Return the tube with the remaining sample to the original box and carefully record the position of each sample in the 96-well plate.
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							* This process is best performed by teams of two with one person transferring the samples and the second recording the positions.
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?. [Silane Bead preparation (performed during swab sample thawing)]
Silane beads (stored at 4C) are washed and resuspended according to the following method (performed outside the flow hood):
?. [Silane Bead preparation (performed during swab sample thawing)]
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).
?. [Silane Bead preparation (performed during swab sample thawing)]
Concentrate beads to the tube wall using a DynaMag-2 magnet; remove bead storage suspension media.
?. [Silane Bead preparation (performed during swab sample thawing)]
Wash 1X with 1 mL of RLT-plus buffer, and remove as in the previous step.
?. [Silane Bead preparation (performed during swab sample thawing)]
Resuspend in 10 μl of RLT-plus buffer per sample.
?. [Silane Bead preparation (performed during swab sample thawing)]
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).
?. [Silane Bead preparation (performed during swab sample thawing)]
Add 70 μl 100% EtOH per sample (7,770 μl for 96 samples), vortex and keep at room temperature:
?. [Silane Bead preparation (performed during swab sample thawing)]
ABC1Reagentx1 samplex96 samples
  (+15 of excess)2Pre-washed Silane beads10 μl1,110 μl3100% EtOH 70 μl7,770 μl4Total80 μl8,880 μl
ABC1Reagentx1 samplex96 samples
  (+15 of excess)2Pre-washed Silane beads10 μl1,110 μl3100% EtOH 70 μl7,770 μl4Total80 μl8,880 μl
?. [Silane bead-mediated RNA purification]
Vortex the Silane bead-ethanol mixture and pour it into a reservoir.
?. [Silane bead-mediated RNA purification]
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.
?. [Silane bead-mediated RNA purification]
Incubate for 5 minutes at room temperature.
?. [Silane bead-mediated RNA purification]
Move the plate onto a DynaMag-96 side skirted magnet and incubate for 3 minutes at room temperature.
?. [Silane bead-mediated RNA purification]
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.
?. [Silane bead-mediated RNA purification]
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.
?. [Silane bead-mediated RNA purification]
Aspirate the ethanol with an 8-channel vacuum aspirator .* Alternatively use a multichannel pipette.
?. [Silane bead-mediated RNA purification]
Repeat the addition of 150 μl of 80% EtOH and incubate for 30 seconds. * Do not re-suspend the beads after adding the ethanol.
?. [Silane bead-mediated RNA purification]
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.
?. [Silane bead-mediated RNA purification]
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.
?. [Silane bead-mediated RNA purification]
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.
?. [Silane bead-mediated RNA purification]
Incubate at 37C for 3 minutes to increase the yield of the elution.* This step may be skipped if yield is sufficient without it.
?. [Silane bead-mediated RNA purification]
Magnetize the plate again by placing it onto the DynaMag-96 side skirted magnet for 1 minute.
?. [Silane bead-mediated RNA purification]
Transfer supernatant into a new 96-well plate.
?. [Silane bead-mediated RNA purification]
Eluted RNA is ready for the RT-qPCR reaction.