I have access to a large database of paraffin embedded samples and I am interested in making the most of it. According to some literature reviews on the subject, the chances of extracting microRNA seem to be better than longer RNA fragments and DNA. However, I would like to hear the opinion and experiences of those of you who might have tried it before.
Generally the smaller, the better. I use Ambion total nucleic acid recover all kit and it works great. I use it for Taqman gene expression and Luminex studies and get good results. Of course you won't always have every sample giving great results but that's FFPE. Try evaluating you RNA on a bio analyzer if you have access to one. I find that in some applications even a low RIN number sample will yield surprisingly good data.
Teresa
In my hands... PFE tissues are not a good source of mRNA, but they are a wonderful source for small RNAs including miRNAs... miRNAs are extremely stable because of their size, so move on. Let me know if I can help.
Good luck.
I have experience of excellent results extracting microRNA from FFPET using commercially available kits. I have compared two leading kits - Roche and Life Tech and experience no difference in microRNA expression (measured by TaqMan qRT-PCR) when comparing total RNA extracted using each kit. I also compared microRNA expression in FFPET samples prepared from 2004 untill 2009 and there was no deterioration in expression over FFPET storage. Finally, the FFPET samples I tested were from 5 different hospitals with different FFPET preperation protocols, and there was no difference in microRNA expresssion between hospitals. It seems that the very small size of the microRNAs makes them very stable and not prone to degradation during the fixation and embedding process.
Generally the smaller, the better. I use Ambion total nucleic acid recover all kit and it works great. I use it for Taqman gene expression and Luminex studies and get good results. Of course you won't always have every sample giving great results but that's FFPE. Try evaluating you RNA on a bio analyzer if you have access to one. I find that in some applications even a low RIN number sample will yield surprisingly good data.
Culpin: It is interesting to note that miRNA derived from FFPE samples is 'sturdy'. I was wondering if this will differ from tissue to tissue. In this regard, would you mind telling what kind of tissue have you analyzed? We are planning to validate miRNA expression in FFPE mouse pancreas. It is notoriously difficult to isolate functional RNA from pancreas and we circumvented this by storing pancreas immediately in RNALater. However, FFPE pancreas was fixed in 10% buffered formalin prior to paraffin embedding. I am wondering if this is sufficient to keep the miRNA intact.
Hi Sundararajan. I have only analysed diffuse large B-cell lymphoma (DLBCL) biopsies and have no experience with pancreas I'm afraid. The DLBCL biopsies are comprised of mixed cell populations and malignant as well as non-malignant cells, so it is interesting that microRNA expression is comparable between the FFPET vs. fresh-frozen sections. Depending on how much sample material you have to spare, I think it would be interesting for you to do some simple comparisons of microRNA expression by qRT-PCR between matched fresh vs. FFPET mouse pancreas sections?
I also use the Recover All kit from Ambion and we got excellent results with TaqMan probes and Real-time PCR but does anyone have an idea about using recovered miRNA for the Affymetrix Genechip expression analysis? what about the quantity required ?
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