On Tuesday, April 16, 2013 12:48:56 PM UTC-4, Pow Joshi wrote: > On 16 April 2013 00:00, DK <[email protected]> wrote: > > > > > In article <[email protected]>, Pow Joshi < > > > [email protected]> wrote: > > > >Dear All, > > > > > > > >I have some cells fixed in 4% formalin (neutral I believe). I was > > > wondering > > > >if anyone has tried any RNA extraction on such cell/tissue samples, and if > > > >you have any wisdom including kits, tricks, solutions, problems that you'd > > > >share with me. I would appreciate it hugely. > > > > > > What's the downstream application? How old are fixed samples and > > > how were they stored? > > > > > > I have next to zero experience with what you are facing and I am > > > mostly curious as to what the real answer is but my guess is that > > > almost all of the RNA (say, 99%) is not recoverable. > > > > > > > > > Well, they have kits for FFPE samples that claim very high recovery. But I > > have never used them and didn't know the details except what's written on > > the kit. I would love to do some real time pcr on the samples. These cells > > were fixed in 2-4% formaldehyde, and kept in the refrigerator 4-8 deg C, > > for about 6mo to an yr. I may have some samples that were fixed for 15 > > min, spun down and re-suspended in PBS for storage. So the formalin > > exposure was minimal. > > yes, I too believe it may be difficult to recover any RNA from them. > > However, I am willing to give it a try if I have some extra information on > > the method(s).
This paper has some tips: http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001261 In your case, since your samples were stored cold for a year or less, you may be ok. (A bigger problem is retrieval of RNA from archival samples) Be advised that the RNA will likely look degraded by analysis by bioanalyzer or other methods, and even "intact" looking RNA may not amplify well because adducts on the RNA inhibit the progress of polymerases. Keep you amplicons small (which will likely be so if you are doing real time) and, if you prime with oligo-dT, near the 3'end. Most of the commercial FFPE RNA isolation kits will include an incubation step in Prot. K in slightly alkaline buffer, which are thought to help remove protein cross-linked to the RNA. Nick -- Nick Theodorakis _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
