TITLE: Sars-CoV2 RNA purification with homemade SPRI beads for RT-qPCR test
AUTHORS: 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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div><div class = "text-block">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.</div></div>

[STEPS]
?. [SPRI viral RNA Cleanup]
Sample dilutionStart 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
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
?. [SPRI viral RNA Cleanup]
Bead BindingAdd 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 foamingIncubate   at  , periodically mix by pippetingMagnetize until solution clears completely (~  ) to make sure you do not loose beadsAspirate the supernatant slowly to avoid disturbing the magnetized beads and discardIncubate  at , periodically mix by pippetingMagnetize until solution clears completely (~ ) to make sure you do not loose beads
25 °C
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
?. [SPRI viral RNA Cleanup]
80% EtOH WashAdd  of 80% EtOHMove the plate from one side of the magnet to the other to immerse the beads in the wash solutionWait for  Remove the wash solution while the plate is on the magnet without disturbing the beadsRepeat 80% EtOH wash (steps 3.1 - 3.4)Perform an extra aspiration of the residual EtOH with a fine tipWait for Remove the wash solution while the plate is on the magnet without disturbing the beadsRepeat 80% EtOH wash (steps 3.1 - 3.4)Perform an extra aspiration of the residual EtOH with a fine tipRemove the plate from the magnet and air dry the beads to remove traces of EtOH (~minutes at , until beads are dry by eye inspection). Avoid overdrying the beads.
120 µl
30 °C
?. [SPRI viral RNA Cleanup]
EluteAdd 10mM Tris pH 8 (for 56ul beads   ) at  - Resuspend the beads fully in the elution buffer by pipettingIncubate for   to allow full RNA elutionPlace the plate on the magnet and wait for the solution to clear (~  )Keep on magnet, and transfer the supernatant to a new plate without disturbing the beadsIncubate for  to allow full RNA elutionPlace the plate on the magnet and wait for the solution to clear (~ )
20 µl
10 °C
22 °C
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 beadsShaking the plateMixing by up-down pipetting about 0.5mm above the bottom of the well