TITLE: Kit-free automated RNA extraction for SARS-CoV-2 testing
AUTHORS: Efthymios Fidanis, Maria Greco, Amelia Edwards, Margaret Crawford, Laura Cubitt, Sophia Ward, Robert Goldstone, Nnenna Kanu, James MacRae, Michael Hubank, Jerome Nicod

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Purpose of examination / Clinical relevance</span></div><div class = "text-block">At the end of 2019, several pneumonia cases were reported in Wuhan, China and the pathogen was confirmed as a new viral strain. World Health organization has named the newly identified coronavirus as 2019-nCoV, also known as SARS-Cov-2. The disease developed into a dangerous pandemic, posing major challenges to the NHS. Although more research is necessary to better understand the virus, in response to the emergency, simple and rapid testing is essential to identify the virus in infected individuals. This will aid the implementation of efficient interventions to contain the spread, and distinguish healthcare workers who have been infected, and are required to self-isolate, from those showing similar symptoms but which are not 2019-nCoV associated. The latter category may continue to work, alleviating stress on hard-pressed healthcare resources. 2019-nCoV is an RNA virus, and the diagnostic tests detect viral RNA in swabs from patient airways using a reverse transcriptase PCR assay. Samples are submitted to HSL, an accredited reporting laboratory, and transferred to the Crick for testing. The first step of the process is sample receipt at the Crick. This SOP describes the extraction of RNA from the inactivated sample.</div><div class = "text-block"><span style = "font-weight:bold;">Principles of Examination</span></div><div class = "text-block"><span>This procedure involves extraction of COVID-19 RNA from </span><a href="https://www.protocols.io/private/E84AE35681AE11EABF5D0242AC110002" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">inactivated virus samples</span></a><span> that have been </span><a href="https://www.protocols.io/private/0B4B016082F611EABF5D0242AC110002" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">aliquoted into plates</span></a><span>. This protocol follows a method developed in-house that is automated on the Biomek FX liquid handling platform. All reagents and buffers prepared in house are batch tested and certified.</span></div></div>

[STEPS]
?. [Biomek FX Setup]
Open the Biomek software, and select the program “RNA Extraction vFINAL” which is located in the “Covid-19” project:
RNA extraction automated on Biomek FX using IN HOUSE protocol.This protocol descibes the processing of one 96-well plate (93 samples and 3 controls).[Note: At the Crick we have two platforms with slightly different decks (FX1 and FX2) and this protocol descibes setup on FX2]
?. [Biomek FX Setup]
Click on “Instrument Setup” as shown below to view the deck layout:
?. [Biomek FX Setup]
Place 4 boxes (lid off) of 250 μl filter tips (green boxes) as shown above.
?. [Biomek FX Setup]
Place 3 boxes (lid off) of 50 μl filter tips (pink boxes) as shown above.
?. [Biomek FX Setup]
Place an Axygen Deep well plate in position P12.
?. [Biomek FX Setup]
At the bench, prepare Beads, Binding buffer/bead mix, PE Buffer, TET Buffer, Sample Plate, Elution Plate and Magnetic Plate as follows:
?. [Beads clean-up]
Take a 1.5 ml tube of pre-aliquoted silica beads () from the fridge and vortex well.
550 µl
We routinely use Silica Magnetic Beads from G-BioScience (see Materials) but we have also tested and validated SeraSil Mag 400 beads (GE Healthcare/Cytiva) as a potential substitite.
?. [Beads clean-up]
Record beads manufacturer and LOT number.
?. [Beads clean-up]
Place on the single tube magnet for  or until supernatant is clear.
?. [Beads clean-up]
Remove and discard supernatant (into the tip waste bin).
?. [Beads clean-up]
Remove tube from magnet and add . Resuspend the beads by vortexing.
[TET]
?. [Beads clean-up]
Pulse spin the tube and place on the magnet for  or until supernatant is clear.
?. [Beads clean-up]
Remove and discard supernatant.
?. [Beads clean-up]
Repeat bead washing for a total of 3 times.
?. [Beads clean-up]
Add . Resuspend the beads by vortexing.Pulse spin the tube and place on the magnet for  or until supernatant is clear.Remove and discard supernatant. (2/3)
[TET]
?. [Beads clean-up]
Add . Resuspend the beads by vortexing.Pulse spin the tube and place on the magnet for  or until supernatant is clear.Remove and discard supernatant. (3/3)
[TET]
?. [Beads clean-up]
Remove the tube from the magnet, resuspend the beads in , vortex and spin down.
[TET]
?. [Binding buffer/bead mix]
In the Falcon tube containing the  (labelled BB, pre-aliquoted) add the beads prepared..
[binding buffer]
?. [Binding buffer/bead mix]
Mix well by inverting.
?. [Binding buffer/bead mix]
Pour into a reservoir and aliquot  immediately in each well of a 96 deep well Nunc plate, using a multichannel pipette. Place on the deck in position P9.
400 µl
?. [PE Buffer]
Add PE buffer to a new reservoir and aliquot  into a new Nunc plate and place plate in position P8.
600 µl
?. [TET Buffer]
Add TET to new reservoir and aliquot   (from batch certified aliquot) into a new Nunc plate and place in position P15.
[TET]
?. [Sample Plate]
Place the sample plate (seal removed) in position P10.
N.B.: If samples have been temporarily stored, briefly spin in the plate centrifuge.
?. [Elution Plate]
Label an empty Eppendorf LoBind 96-well skirted plate with a pre-printed SPL barcode (e.g. SPL00000) on the front side, and place in position P16.
?. [Magnetic Plate]
Place the Alpaqua Magnum FLX magnetic plate in position P13.
?. [Final Setup]
The final set up should look like the photo below:
?. [Final Setup]
Using the laptop beside the FX, push the samples through the Extraction step in ClarityLIMS (refer to ‘COVID-19 LIMS walkthrough’ document on COVID-19 sample handling landing page).
?. [Final Setup]
Make sure you switch ON the Inheco (blue box next to the FX monitor).
?. [Final Setup]
To START the program press the Run button (green triangle) located in the software.
The robot carries out the following automated protocol1. Transfer 150 μl of samples to 400 μl (2.6x Vol) of Binding Buffer (BB) containing the Silica beads and mixe 10 times by pipetting up and down2. Move the plate on the Shaker for 5 min3. Move plate to position P8 and mix 20 times4. Move plate to Shaker for 2 min5. Move plate to P8 and incubate for 3 min6. Move plate to Magnet for 5 min7. Remove supernatant8. Move plate from magnet to position P8Wash with PE (1)9. Transfer 180  μl of PE Buffer in each well of the Bead Mixture plate10. Move plate to the Shaker for 20 sec11. Move plate to the Magnet for 60 sec12. Remove supernatant13. Move plate from the magnet to position P8Wash with PE (2)14. Transfer 180  μl of PE Buffer in each well of the Bead Mixture plate15. Move plate to the Shaker for 20 sec16. Move plate to the Magnet for 60 sec17. Remove supernatant18. Move plate from the magnet to position P8Wash with PE (3)19. Transfer 180  μl of PE Buffer in each well of the Bead Mixture plate20. Move plate to the Shaker for 20 sec21. Move plate to the Magnet for 60 sec22. Remove supernatant23. Move plate from the magnet to position P824. Wait for 2 min (drying)Elution25. Transfer 40  μl of TET Buffer to the Bead Mixture plate and mix 10 times26. Move plate to the Shaker for 5 min27. Move plate to the magnet for 60 sec28. Transfer 35  μl from the Bead Mixture plate to the Eppendorf twin.tec plate
?. [Final Setup]
When then program is complete, remove the Eppendorf 96-well skirted plate from the deck, and seal with a Thermo Adhesive PCR plate seal.
?. [Final Setup]
Continue with the qPCR RNA Transfer or place in the  until it is used for qPCR setup.
-80 °C
?. [Archiving]
Archiving of Inactivated viral samples would have already been performed after sample plating. For archiving the stock RNA plates using the app in Clarity LIMS, a database table is updated with the information.
?. [Archiving]
This results in an entry appearing on the dashboard to let the team know the samples are ready to be archived:
?. [Archiving]
An archiving app then displays the boxes and plates that are waiting to be archived, and gives the team the chance to enter the storage information:
?. [Archiving]
Once logged, the record disappears from this screen, and the archiving information will appear in the archive search app: