[admin]

Another great contribution from the wider BOMB community:

• This topic was modified 4 years ago by admin.
• This topic was modified 4 years ago by admin.

[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.

[TimH]

Hi Bradley,

Many thanks for the message and sharing your protocol. Do you have a way to show/test inactivation of the virus? Commercial AVL is ineffective at inactivation of Ebola (https://journals.sagepub.com/doi/pdf/10.1177/1535676017703383), so please take this into consideration if/when you start working with actual virus. Be safe. You could consider trying a GITC buffer with some detergent as this may improve inactivation (e.g. our GITC lysis buffer from the ‘TNA’ protocol #6.1 as below):

GITC 4 M 23.64 g
Tris HCl pH 7.6-8.0 50 mM 2.5 ml of 1 M stock
Sarkosyl 2% 1 g
EDTA 20 mM 2 ml of 0.5 M stock
Antifoam (optional) 0.1 % 50 μl
adjust pH with HCl to 7.6-8.0 and adjust the volume with water to 50 ml

This protocol (TNA #6.1) also has also been used routinely with SpeedBeads as you propose, however, we prepare them in only TE (i.e. no salt), and at 1:50 dilution from the original stock, following 2x TE washes to get rid of sodium azide and other components of the storage medium.

 

Best wishes,

Tim H

 

[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.

[Rob Page]

Thank you so much for sharing your protocol and I’m glad to hear it worked with the spiked in RNA control. Similar to you, we have access to samples that have already been tested with commercial kits in diagnostic labs. We will compare and feedback here how it goes.

For the inactivation, we cannot propagate the virus in our lab but have a collaborator that can and is willing to test various lysis buffers for inactivation. Our plan was to use to one from the TNA protocol which includes detergent, but I can ask if they have capacity to also test the inactivation with the lysis buffer in the RNA specific-protocols. I will let you know what they find once I have the data.

[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

 

[Emily Junkins]

Hi everyone,

I am working with Bradley Stevenson and wanted to ask a few more questions. We are still using the above procedure with the Sera-Mag prepped beads without PEG. I saw another thread here about bead clumping (specifically carboxylated beads) that suggested it could be both a GTC issue and/or a nucleic acid concentration issue.

1. Your 8.2 protocol uses 40uL beads with 560uL of sample+buffer, when we add 40uL of beads it is to over 1 mL of sample+buffers because of the viral transport medium and lysis buffer (we cant spin down). The thread mentioned above seems to hint at the amount of beads won’t help that issue, but it is something I think is worth a try. Any pointers about bead amounts relative to sample volume?

2. As we worked through out protocol, we had issues towards the end with beads sticking to plastic (low-bind eppendorf) and not interacting with the magnet. We think this could be due to high concentrations of nucleic acids. This is problematic for us since we are trying to set this up for liquid handling systems that won’t be able to scrape beads like we can manually. Have you experienced this?

3. I would like to try your protocol in its entirety but using our prepared carbox beads. Have you encountered any issues adapting this or foresee any when switching to automation?

I realize that we are asking questions about completely different protocols other than the BOMB 8.2. I am hoping you or anyone may have some insight into what might be best for RNA recovery and automation using a magnetic, kitless approach for COVID-19.

Thanks,

Emily

[admin]

Hey Emily,

Unfortunately I am not as savvy with the molecular processes behind what is happening as other members of BOMB team who will hopefully chime in.

1. Using the 8.2 protocol I have encountered clumping of beads in samples I knew were a bit too concentrated (contained too much tissue). However this never led to any problems down the line.  I can imagine that efficiency in these samples is reduced but that never mattered for my purposes.

2. I have used a relative large selection of magnetic racks and can say that they vary hugely in magnetic strength, which in turn has a profound effect on the time it takes for the beads to migrate/clear.  However, truly insufficient interaction with the Magnets is something I have encountered only when using less than ideal combinations of magnetic racks and tubes/plates. In other words it was always  a matter of physical proximity between the inside of the vessel and the magnet surface.

3. Concerning automation the biggest problem I see (not considering the sticky issue) is the exact position of the pipette tip relative to the magnetically congregated beads. With different racks/magnets the beads might form a ring around the bottom a line on the wall or even two dots.  All of which is fine as long as you can adjust the position of the tip removing buffer precisely.

Cheers

Tim M

 

 

[Phil Oberacker]

Hi Emily,

1. As Tim mentioned correctly, clumping of the beads is in my experience a sign that the beads reached their capacity limit. Is it only happening during the washing steps, or already before? I recently uploaded an update on bead capacity and clumping behaviour (26.02.20) in the project log of the BOMB project on Researchgate. Maybe this can help you figuring out the best amount of beads: https://www.researchgate.net/project/Bio-On-Magnetic-Beads-BOMB
2. Indeed, we also never encountered this issue before (even if we have the clumps/flakes when the beads are fully loaded) and I agree with Tim that either the magnets are not strong enough or too far away from the beads. What liquid handling system do you use?
3. Some people had issues in the past, when they were using some of our protocols with commercial beads. However, the COVID-19 RNA isolation protocol we uploaded today also uses commercial Sera-Mag SpeedBeads with a carboxyl coat: https://bomb.bio/wp-content/uploads/2020/04/SARS-CoV-2-RNA-purification-from-nasal-swabs_BOMBv2.pdf
   For automation I don’t see any troubles. As Tim mentioned, for automation it’s better to use ring magnets, as the beads are distributed in a more convenient way for the robot. However, also here it really depends on the magnet strength. I recently worked with a magnet plate from Alpaqua, which is specifically designed for liquid handling machines, but the magnetic force of this plate was far lower than what the magnets used in our homemade plates were able to exhibit. It still worked, it just took longer (a few seconds vs. almost a minute).

If you have any more questions, don’t hesitate to ask 🙂

Cheers
Phil

[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]

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

[Kylie Drake]

Emily,

Ideally, collecting straight into GITC will solve the volume problem. But we had to make do with current practice in our laboratory. Who is adding your swabs to VTM? If it isn’t happening at the clinic, consider a move to GITC.

One way to reduce your input volume is to increase the molarity of the GITC lysis buffer. That’s why the COVID-19 protocol uploaded here uses 6M GITC. If you maintain a 2:1 ration of VTM:GITC and a 5:4 ratio of beads+GITC+VTM:isopropanol you should be able to reduce your input volume. just be careful that the 6M GITC doesn’t precipitate.

We started off in a 2.2mL deep well plate and the volume was too high to facilitate settling of the beads. The ability of the magnet to pull down the beads decreases exponentially the further away from the magnet you are. So unless you have an automation system with a magnet that surrounds your entire well (Abbott m2000), or has a magnet that can be immersed in the well (Kingfisher), you’ll have trouble getting the beads to pellet with such a large volume.

One trick that worked is to “clarify” the solution by slowly pipetting the volume past the magnet. Bringing the beads closer to the magnet will allow them to be “captured”. This works to an extent, but we still found that it took upwards of 45 minutes to pellet the beads in a large volume (>1mL). Which is ultimately why we settled on a 0.8mL plate and a smaller volume. You can then use either a ring magnet or something like this the Invitrogen™ AM10027 magnet. The AM10027 is what we’re using at Canterbury Health Labs.

To date we haven’t encountered any issues with clumping of the beads. Although we’ve yet to try extracting from sputum samples. I wouldn’t think there were enough cells in a throat or nasopharyngeal swab to cause clumping.

Good luck!

Kylie

[Phil Oberacker]

Hi Bradley,

getting RNA from yeast shouldn’t be a problem. With the original paper we aslo published a protocol to isolate TNA from Saccharomyces (#6.5). We basically just break up the cell wall with a lyticase digest and go on like in any other BOMB TNA extraction. That also means, that allows you to basically switch to one of our RNA protocols (#8.1, #8.2) in between and add a DNase I digest after the first round of washing.

Cheers
Phil

PS: Couldn’t agree more 😀

[Emily Junkins]

Hi all,

We are about to try the new BOMB COVID-19 extraction, this is nearly identical to how we prepare for NGS and have good results, so I am very excited to get it working and automated (we’ll share the programming for the opentron system once it gets going). I do want to share though that we added 80mM of DTT to the lysis buffer with GTC before use and saw no more clumping if that is help to anyone on any other protocols.

Thanks for all of the insight and getting this protocol shared!

[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

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