On 17 April 2013 14:03, Pow Joshi <[email protected]> wrote: > > > On 17 April 2013 10:54, Nick Theodorakis <[email protected]>wrote: > >> 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. >> >> 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 >
On a related note, I read the Plosone paper that Nick sent, the proteinase K and heating to 65-70 deg C is the method to digest the bound proteins and break the methylene crosslinking bridges. I believe it is done in that order, protease then heat. I was wondering if one could potentially do it in the reverse, heat first then add a smaller amount of protease? and would that lead to a better yield ?.... has anyone tried any such combinations, and theoretically if there could be problems such as RNA hydrolysis or any others.... I'd appreciate anything you may have to add to this discussion! Thank you, Pow >> _______________________________________________ >> Methods mailing list >> [email protected] >> http://www.bio.net/biomail/listinfo/methods >> > > _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
