@alejandrocaro
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Dear Rob,
we try adding half and double the amount of bead solution and did not find much difference in the final Ct. One thing that really helps us was to increase Ct was the incubation of 10 min @ 60 C with Proteinase K.
We have not tested the addition of mucolytics to the extraction, but Alexander James Phillips suggested several reagents that might work https://testingmethods.<wbr />crowdicity.com/post/3193060 .
The following paper also tested the effect of several conditions and concentrations, that might be also adapted.
He, H., Li, R., Chen, Y., Pan, P., Tong, W., Dong, X., … & Yu, D. (2017). Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections. Scientific reports, 7, 45199.
Best,
Alejandro
<h3 class=”iw”></h3>Dear All,
we have started diagnosis at my institution (AGROSAVIA, Colombia) two weeks ago and we have been running in parallel both the column extraction kit and your protocol for around 20 samples. We have noticed that the RNaseP gene amplifies at the same CT (human RNA extraction internal control), however, the RdRP gene (SARS-Cov2) amplifies around three CT after. Another observation is that whenever there is Blood in the samples we failed to amplify the RdRP gene, beads seem to get attached to the blood. For these samples, we try a 1/4 dilution and got RdRP to amplify.
To improve the RNA extraction for the virus, we plan to increase 50% and 100% the volume of beads in the protocol.
We would like to know if you have any suggestions, for those samples with blood and to increase the extraction efficiency of viral RNA. Do you think a higher RNA extraction efficiency can be achieved by adding the RNA binding buffer (protocol 8.2 with Gu-HCl)?
PS. I know Oscar Javier Ortega, who was one of my students at a class at UNIANDES, and who has been of enormous help to start this protocol.
Best,
Alejandro
