On 17 April 2013 10:54, Nick Theodorakis <nick.theodora...@gmail.com> wrote:
> On Tuesday, April 16, 2013 12:48:56 PM UTC-4, Pow Joshi wrote: > > On 16 April 2013 00:00, DK <d...@no.email.thankstospam.net> wrote: > > > > > > > > > In article <mailman.259.1366062468.10461.meth...@net.bio.net>, Pow > Joshi < > > > > > pow.jo...@gmail.com> 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. > > Nic > Nick Theodorakis > Thank you, both, Engelbert, and Nick. I will keep these in mind, and look at the Plosone paper as well. It woudl be awesome if the samples can be used for real time pcr assays ... Pow > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
