@robertpage
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Thank you very much for your reply Alejandro. It’s interesting that Proteinase K 10 mins @ 60 degrees should make a difference to the RdRP CT and not the RNaseP. Do you think it is simply breaking down the mucus/proteins to make the RNA more accessible to the beads? I will give it a go with our protocols tomorrow and let you (and everyone else here) know.
Can I ask why 60 degrees? Is this based on the scientific reports paper you linked that shows lower CTs at higher temperatures (although the authors are not clear which steps of their protocol are at higher temperatures). It seems to me that this temperature is optimal for RNase activity (although the GITC and Proteinase K will protect against this).
Hi Alejandro,
I am working at King’s College London and Guy’s Hosptial to set up and validate as many RNA extraction protocols for SARS-CoV-2 as possible, including several silica based methods we have developed in house. We are seeing the same thing as you described. When compared to commercial kits, our RNaseP CTs are exactly the same, but we lose 2-3 CT on our CoV primer-probes (CDC N1 & N2).
Interestingly we do not see this effect if we dilute our samples 1:10 (although we lose sensitivity by all methods), so our working hypothesis is that gDNA (larger) may be outcompeting the viral RNA (smaller) for binding where there is a high overall concentration of nucleic acid. The RNaseP primers will amplify gDNA, which is why there is no loss of signal.
I can see from your paper that you managed to solve this problem. You mentioned that you planed on increasing the amount of beads? Did this work or was there any other ticks that made the difference.
Any help very much appreciated!
Cheers,
Rob
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.
Hi,
I am also working on COVID19 diagnostics, based in London. We are going to try to use the GITC lysis RNA extraction protocol on our samples because sourcing commercial kits is starting to become a serious issue. If we can make our own then hopefully that would be much better!
I wondered what volume of lysis buffer you would recommend adding to our samples? We cannot centrifuge to pellet the virus, so we have to start from a suspension of the virus in transport media (HBSS) the swabs come in. Or we could make a more concentrated version of the lysis buffer to dilute to 1x in the transport media? I know we need to keep the lysate:isopropanol:bead ratio consistent. Any help would be greatly appreciated.
