5 replies to this topic

[aliquot]

I've been trying to extract viral RNA from tissue culture supernatant using the TRIzol LS reagent.  I've followed the protocol exactly and gotten next to nothing in return.  I never see a pellet and the spec reads are zilch.  I re-cleaned all of my equipment and replaced all of my solutions to ensure that everything is RNase free, nothing.  I tried a protocol online from UPenn that is a mixture of the TRIzol and RNeasy kit procedures, nothing.  I contacted Invitrogen and they said to try diluting it in PBS to lower the serum concentration, nothing.

I know there is virus in these samples, according to plaque assay upwards of 10^9 PFU/mL.  The only thing I have not tried is a carrier like glycogen.  Is glycogen really going to help me that much or should I just try a product like this: https://www.roche-ap...Id=3.6.8.54.1.1 ?  I'd like the RNA to be usable for RT after extraction.

Any help is appreciated.  Thanks.

Edited by aliquot, 27 December 2011 - 10:20 AM.

[akhshik]

is it just a supernatant from tissue culture or you have plant cell lysate?
culture media and plant surface are full of RNase and autoclave do nothing about it as you know only a few materials such as DEPC could treat RNase,

I had a friend in lab she freez culture media with cells and then extract as fast as she could to get some RNA!!!

[aliquot]

I said nothing about plants in this post.

It is supernatant from mammalian tissue culture, I know there is virus in there, I've done plaque assays using these samples, therefore there must be RNA in there since it is an RNA VIRUS.  The viral proteins protect the RNA from degradation in the media (otherwise it wouldn't be able to infect anything obviously).  Then, the TRI reagent should inactivate any RNases in the media when I add it, yes?

[leelee]

A few questions.....

Have you (or someone else in the lab) done this before, and its now not working? Or is this the first time?

The plaque assays you've previously performed- are they from the supernatant only? Or do you typically lyse the cells also?

[aliquot]

First time anyone in the lab has done this.

Supernatant only, but when I harvest virus most of the cells have died anyway, so I'm sure there are cellular proteins in there.  I collect supernatant, spin down any cellular debris, and aliquot the infected media.

[WSN]

I don't know how much does 10^9 PFU/mL mean for RNA, might not much. I would suggest not to spin at all, viral particles are pretty big themselves, you could lose a lot of them to debris, especially those that attach to cytomembrane. Trizol can handle high protein and lipid load.  And the carrier really help in recovery of trace amount of RNA, so use some. Good luck!