[Bradley Stevenson]

p.s. You can change the name of this thread/channel if you want to too!

[Bradley Stevenson]

Hi everyone,  sorry for the silence. We have essentially moved over to the bomb.bio protocol, no NaCl in our bead prep (just Sera-mag carboxy beads in TE), getting rid of our salt-containing wash step, and using 90 uL of beads. I will get some details and add our information to the database. Also, we just went ahead and engaged the magnet the entire time, without loss of efficiency.

Stay safe,

Brad

[Bradley Stevenson]

Just an update, Emily Junkins ran both the Hillbilly protocol and the BOMB.bio protocol in parallel today on the same samples. She was able to detect positive and negative samples with 100% accuracy. Both extractions came up with similar extraction efficiencies but we will compare the data and re-run in the near future. Also, I successfully set up an RT-qPCR reaction in a lab not my own and on an instrument I have never used before (mostly, as I didn’t make enough mastermix). PI’s must flex when this happens!

Cheers,

Brad

[Bradley Stevenson]

BTW, I cannot wait to solve important problems with open science, let’s rip this s*** open and get important science done.

[Bradley Stevenson]

Hey Phil,

Thanks for your input and your great protocol. We have essentially arrived at what you outline in your protocol but are awaiting results (from actual patient samples). One thing that we have seen that matters is the use of carrier RNA. Our choice has been hybridoma cells that are available where we are (IMMY, immy.com), but we will need to figure out a more readily available source of carrier RNA (Saccharomyces?).

[Bradley Stevenson]

Hey Rob,

Thanks for the link. I wondered how a lysis step that makes the viral RNA available for amplification would not be inactivating the same virus. I suspect that high titers are to blame. I had originally thought we would use a heated incubation of the lysis step until our colleagues got positive results without it. I think I am adding it back in!

“…Since infectivity was detected with these 2 viruses, a separate 10-minute incubation in lysis buffer at 65C was performed. The disruptive effect of heat incubation on viral envelopes and genomes is well documented,4,5 and as expected, infectivity was not observed when the heat-treated virusbuffer mixtures were subsequently cultured and passaged. These results suggest that an additional heat step should be considered when using these buffers if inactivation is not initially achieved.”

The discussion of the Ngo et. al paper was enough to convince me!

Will let you know how our “real” extractions go and post a cleaner protocol soon.

Cheers, Brad

 

[Bradley Stevenson]

Hey, just FYI. This protocol (with a few minor edits) worked. We ran it using some human hybridoma cells spiked with synthetic RNA specific for the N1 and N2 primers in the CDC test kit. Next, we will run it with samples from both positive and negative tests that the same facility has tested with commercially available kits. If we can get 100% compliance, we will post the protocol with annotation for all to use.

[Bradley Stevenson]

My colleagues have been successful in recovering viral RNA from the transport media using the following protocol and Qiagen RNEasy columns. We are starting there and trying to simply replace the filter cartridge with steps using carboxylated GE Healthcare Speedbeads. We are assuming that binding will be same (unknown) in the lysis and binding buffers in the attached protocol. In other words, I dont think you will need to concentrate your sample.  We are literally sitting down to work on this now with things I moved from my lab. I will let you know how it goes.

Hillbilly Bead Viral RNA Extraction

Viral Lysis Buffer (AVL) – In House – 50 mL Recipe

• Guanidine Thiocyanate – 35.44 g
• Tris Base – 0.61 g
• RNAase Free Water – Bring to Volume 50 mL
• pH 7.3-7.7

 

Carrier RNA

• 1 mL AVL contains 10 ug Carrier RNA
• 1 mammalian cell contains approx. 20 pg RNA
• Add 5 x 10⁵ hybridoma cells per mL AVL

 

Hillbilly Bead Preparation (a.k.a Preparation SpeedBead Magnetic Carboxylate Modified Particles)

• SpeedBead Magnetic Carboxylate Modified Particles (GE Healthcare; 65152105050250)
• NaCl (e.g. Sigma Aldrich,S3014-500G)
• 1 M Tris-HCl, pH 8.0
• 5 M EDTA, pH 8.0
• DEPC-treated water

 

For 50mL of Hillbilly Beads

1. To a sterile 50 mL conical tube, add 2.92 g NaCl with ~40 mL of water, vortex to dissolve. Add 0.5 mL of 1M Tris-HCl and 0.1 mL of 0.5 M EDTA and add water to 50 mL. This is the TE to be used next.
2. Thoroughly resusped particles in stock of SpeedBead Magnetic Carboxylate Modified Particles. Transfer 1 mL to a 1.5 mL microcentrifuge tube.
3. Place beads in rack on magnet and wait until solution is cleared (2-3 min). Carefully remove liquid with pipettor, do not aspirate beads.
4. Add 1 mL of TE (freshly prepared above) and resuspend particles. Place on magnetic rack and allow solution to clear. Carefully remove liquid.
5. Repeat washing beads with TE two more times.
6. Resuspend beads in 1 mL TE and set aside.
7. To a new sterile 50 mL conical tube, add 2.92 g NaCl with ~40 mL of water, vortex to dissolve. Add 0.5 mL of 1M Tris-HCl and 0.1 mL of 0.5 M EDTA and add water to 49 mL.
8. Transfer TE-washed particles to prepared solution and mix. This is the Working Stock of particles (2% SpeedBead Magnetic Carboxylate Modified Particles, 1M NaCl, 10 mM Tris-HCl, 1 mM EDTA).
9. Store at 4oC for up to 3 months. Mix well before each use.

 

Wash Buffer 1 (AW1) – In House

• 6M Guanidine HCl – 19 mL
• 95% EtOH – 25 mL
• pH 4.5-5.5

 

Wash Buffer 2 (AW1) – Use Promega RNA Wash Solution

 

Elution Buffer (AVE)

• RNAase Free water

 

Procedure

1. Pipet 560 uL prepared Buffer AVL containing carrier RNA into a 1.5 mL microcentrifuge tube.
2. Add 140 uL of sample (in Viral Transportation Medium). Mix by pulse-vortexing for 15 s.
3. Incubate at Room Temperature (15-25oC) for 10 min.
4. Briefly centrifuge to remove any liquid from inside cap.
5. Add 560 uL of 95-100% Ethanol. Mix by pulse-vortexing for 15 s.
6. Add 40 uL of Bead Solution. Mix by vortex for 5 min.
7. Place tube on magnet to pellet beads (2-5 min or until solution is cleared).
8. Remove buffer, being careful not to aspirate beads
9. Remove tube from rack, add 560 uL of Buffer AW1. Mix by pulse-vortexing for 15 s.
10. Place tube on magnet to pellet beads.
11. Remove buffer, being careful not to aspirate beads.
12. Remove tube from rack, add 500 uL of Buffer AW2, mix by pulse vortexing.
13. Place tube on magnet to pellet beads.
14. Remove buffer and repeat wash with AW2.
15. Dry beads at 50oC for 15 min to remove traces of ethanol.
16. Add 60 uL Buffer AVE. Mix by pulse-vortexing for 15 s. Incubate for 5 min. at room temperature.
17. Place tube on magnet to pellet beads.
18. Transfer RNA-containing solution to a clean tube.

[Bradley Stevenson]

This protocol seems very useful for my desire to develop an open source, do-it-yourself, protocol for extracting COVID-19 viral RNA from samples. Every time another commercial kit is approved, it is backordered for an unknown amount of time. I am a University professor who is shut out of his own lab that happens to know the CEO of a local biotech company. They are trying to ramp up the capability to become a testing lab for COVID-19 samples. We are in Oklahoma, and are about 1-3 weeks behind the curves on the East and West coasts. Given what shortages they are experiencing now, by the time we need to be able to test widely (weeks ago in my opinion), the resources to do so will be even more scarce. We are hoping that if we can obtain the raw materials for the extraction of viral RNA using GITC and magnetic beads, we could adapt this to multiple automated platforms. We are most interested in the Opentrons platforms, as we are 100% interested in sharing our successes with the worldwide community so that everyone can benefit. Let’s democratize testing for COVID-19 because we are all in this together as a species.

[Author]

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