Can you concentrate RNA in Plasma? - (Aug/08/2006 )
I’m working HIV Virus from human plasma collected in EDTA treated vacutainers. Some of these patients have very low viral load, <50 copies/ml, after treatment. Most of these samples I can’t amplify them.
Someone suggested I should centrifuge the sample for 1 hour at 17,000 rpm in 4°C. discard 3/4 of the supernatant and resuspend it, before extraction and RT-PCR. Can you really concentrate viral RNA this way?
If not, what can I do?
Yes, you can. I was at a conference recently and a poster there provided the results about ultracentrifugation for one hour, and then purifying the RNA and doing the RT-PCR.
It's in the abstract book of the "4th European drug resistance workshop". Abstract 63, but Stone et al, working for GSK. The performed high speed centrifugation (50.000g) and standard lysis and extraction. Worked well for them on short pieces.
We're doing it ourselves here (not 50.000g, but 20.000g, but I think you can try @ 17.000) also and having higher sensitivity than other extraction methods.
I'll try it. Thanks
I notice that is a layer of white stuff on top off the tube after spinning it. And when I start my extraction, I notice it start to bubble up... looks like the foam on beer. Shouldn't the RNA pallet down just like DNA?
Never happened to me, but your RNA should pellet down, but take in mind that you might not see the pellet as there is too little RNA to visualise (definately when starting with low viral load samples).