TITLE: Step 2: RNA extraction and RT-qPCR
AUTHORS: Peter H L Krijger, Tim A Hoek, Sanne Boersma, Lieke I P M Donders, Maaike M C Broeders, Mark Pieterse, Pim W Toonen, Ive Logister, Bram M P Verhagen, Marjon J A M Verstegen, Thomas W van Ravesteyn, Rene J T M Roymans, Francesca Mattiroli, Jo Vandesompele, Monique Nijhuis, Stefan Meijer, Anton van Weert, Edwin Dekker, Fred J Dom, Rob Ruijtenbeek, Lieven B J van der Velden, Jeroen H B van de Bovenkamp, Martijn Bosch, Wouter de Laat, Marvin E Tanenbaum

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">STRIP is a start-to-end streamlined and automated procedure for COVID-19 testing, centering on a single Tecan Fluent liquid-handling robot that can process over 14,000 samples per day. </div><br/><div class = "text-block">Key features of the customized Tecan Fluent robot are (1) on-board 1D and 2D barcode scanners, (2) an automated tube decapper, (3) two robotic arms for simultaneous processing of different procedural steps, (4) a newly-designed spin vessel to keep magnetic beads in solution and immediately transferable to 384-well plates, (5) a built-in magnetic deck and a 384-channel pipetting head for rapid RNA isolation, (6) a heating device for fast drying of RNA prior to elution, (7) a built-in plate sealer and (8) a plate storage system to allow processing of multiple sample plates in a single run (See Materials).</div><br/><div class = "text-block"> Here we describe the RNA extraction and RT-qPCR protocol as currently applied in STRIP. </div></div>

[STEPS]
?. [RNA extraction and qPCR setup]
Combine in 1 micronic tube rack: 1 micronic tube with the positive extraction control (position A1)1 micronic tube carrying the negative extraction control (position B1)1 micronic tube carrying the negative amplification control (position C1)93 micronic tubes carrying swab specimens
?. [RNA extraction and qPCR setup]
Briefly spin the micronic tube racks in an under bench centrifuge ( ,  ) and place in the Tecan Fluent carousel with the barcode facing the user.
Centrifuge: 500 34
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Start the RNA extraction and qPCR setup script to initiate the automated workflow:
?. [Preparation of the Fluent]
Take out 10ml aliquots beads/isopropanol mixture from  storage, vortex and resuspend in 2-Propanol as described in step 11-14 of the bead preparation protocol.Transfer the diluted beads into the spinvessel. Start the spinvessel, this will keep the beads in solution. Beads can be used for 5 days at RT.
4 °C
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?. [Preparation of the Fluent]
Startup the Tecan Fluent.
?. [Preparation of the Fluent]
Refill Nuclease-Free Water in  H2O bath and turn on H2O bath.
80 °C
?. [Preparation of the Fluent]
Refill 70% EtOH in the hydrospeed tank (Note: in future designs the hydrospeed will be replaced with a multidrop).
?. [Preparation of the Fluent]
Place the RNA extraction plates, EtOH plates, plates, 384-well qPCR plates, 125 µl disposable tips, and 50 µl disposable tips in appropriate positions of the stacker.
?. [Preparation of the Fluent]
Check if a sufficient amount of seal is present in the plate sealer.
?. [Preparation of the Fluent]
Check if waste bins need to be emptied.
?. [Preparation of the Fluent]
Prepare qPCR mix in a 50 ml tube wrapped in aluminum foil and place in cooling unit. Note: for each 384-well plate a new 50 ml tube with qPCR mix is prepared. 700 reactions of the RT-qPCR mix is prepared per 384-well plate due to the dead volume of the 50ml tube and tubing.
?. [RNA extraction and qPCR setup]
The 2D barcodes of the collection tubes are scanned using a Ziath Mirage Barcode Scanner.
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A 150 µl RNA extraction plate is placed at the deck.
?. [RNA extraction and qPCR setup]
Micronic tubes are de-capped per tube-rack (96 samples) in the Brooks IntelliXCap 96 Capping/Decapping Unit.
?. [RNA extraction and qPCR setup]
125 µl pipet tips are attached to the MCA96 head. 30 µl bead solution is aspirated, then 60 µl sample is aspirated, and the entire 90 µl is dispensed in the RNA extraction plate and mixed 5x.
?. [RNA extraction and qPCR setup]
The Micronic tubes are recapped and returned to the Tecan carousel.
?. [RNA extraction and qPCR setup]
Steps 11.1-11.5 are repeated for 3 other 96-tube racks, to completely fill the RNA extraction plate.
?. [RNA extraction and qPCR setup]
Incubate for .
?. [RNA extraction and qPCR setup]
The RNA extraction plate is placed on the magnetic deck and incubated for .
?. [RNA extraction and qPCR setup]
The 300 µl waste collection plate is placed at the deck.
?. [RNA extraction and qPCR setup]
The 150 µl EtOH plate is filled up with 150ul 70% ethanol using the Hydrospeed and placed at the deck.
?. [RNA extraction and qPCR setup]
The MCA 96 head is replaced for the MCA 384 head and 384x 125 µl tips are attached.
?. [RNA extraction and qPCR setup]
The supernatant present in the RNA extraction plate is aspirated using the 125 µl tips and transferred to the waste plate.
?. [RNA extraction and qPCR setup]
The RNA extraction plate is placed at the deck.
?. [RNA extraction and qPCR setup]
Using the same pipet tips, 30 µl 70% EtOH is aspirated from the EtOH plate, dispensed into the RNA extraction plate, and mixed 3 times.
?. [RNA extraction and qPCR setup]
The RNA extraction plate is placed on the magnetic deck and incubated for .
?. [RNA extraction and qPCR setup]
Using the same pipet tips, the Etoh is aspirated from the extraction plate and transferred to the waste plate.
?. [RNA extraction and qPCR setup]
Step 11.13- 11.16 is repeated 3x (4x total).
?. [RNA extraction and qPCR setup]
The RNA extraction plate is placed at the Bioshake and heated at  for .
80 °C
?. [RNA extraction and qPCR setup]
The waste plate is sealed and transferred to the waste bin.
?. [RNA extraction and qPCR setup]
The RNA extraction plate is placed at the deck.
?. [RNA extraction and qPCR setup]
The lid from the  H2O bad is removed and using 50 µl 384 pipet tips attached to the MCA 384 head 25 µl H2O is aspirated, dispensed in the extraction plate, and mixed 21x ( total incubation) to elute the RNA.
80 °C
?. [RNA extraction and qPCR setup]
In parallel, 7.5 µl of the multiplex RT-qPCR master mix is added to a barcoded 384-well qPCR plate.
?. [RNA extraction and qPCR setup]
7.5 µl of RNA extracted from the samples (out of 25 µl total RNA) is transferred from the extraction plate to the qPCR plate using the tips to pipet the H2O.
?. [RNA extraction and qPCR setup]
The qPCR plate is sealed.
?. [RNA extraction and qPCR setup]
The barcode of the sealed qPCR plate is scanned and the plate is stored in the robot, ready for RT-qPCR analysis.
?. [RNA extraction and qPCR setup]
The EtOH plate is transferred to the waste bin.
?. [RNA extraction and qPCR setup]
Briefly spin the qPCR plate () and transfer immediately to a Quantstudio 7 thermocycler.
Centrifuge: 500 34, 1 min
?. [RNA extraction and qPCR setup]
The processing of the RT-qPCR output is automated using custom-designed LabTrain software by Bodegro. Positive samples are manually curated by trained professionals. Negative samples that show inhibition of PDV amplification (Cq > 33) are repeated. After manually curation, the results (positive or negative for SARS-CoV-2/COVID-19) are sent digitally to CoronIT (software designed for the Dutch Municipal Health service / GGD). A clinical GGD doctor informs and advises the person who has undergone the covid-19 test about the PCR result.
?. [RNA extraction and qPCR setup]
Run the RT-qPCR program.Time (s)Temp (°C)1x300551x209540x3953058
Time (s)Temp (°C)1x300551x209540x3953058