Hi.
I have formalin fixed tissue and FFPE tissue from the same samples which have been in storage for 1-2 weeks. I am looking to obtain nucleic acids for qPCR, where the target is 80bp. Is there a preference for using tissue just in formalin (10% Neutral buffered) or the FFPE for extraction? Obviously the formalin fixed doesn't required the xylene washing to get rid of the wax so this might be better? Or is the fact that FFPE has been in the formalin for less time more important?
Thank you for any advice.
Hello Teresa Wilson
I would not recommend formalin fixed tissue stored for 1-2 weeks as samples to be used for extraction of nucleic acids for qPCR. The samples that have been fixed immediately with 10% neutral buffered formalin for only up to 1 day or less can be subsequently used in PCR amplification. So, you should go for FFPE tissue that has been in formalin for less time for extraction of nucleic acids.
You may want to refer to the article which I have attached below for more information.
Article Assessment of the quality of DNA from various formalin-fixed...
Best.
Hello Malcolm
So you would say that even though the formalin bonds are still there in the FFPE, the affect of this is less than the affect on the tissue soaking in formalin. The paper is excellent, however it doesn't make this direct comparison. I have not been able to find a direct comparison for these two things. But I can see the comparison between FFPE and fresh tissue is useful.
kind regards Teresa
Dear Teresa
Formalin fixed fresh tissues are never used for RNA extraction. This is a wrong practice, as formalin penetrates into tissues with a rate 1 mm / hour in general depending on its concentration used for fixation and hence after 24 hours the formalin-dipped tissue is no longer useful to generate any good RNA. Especially, the outer parts of tissues affect more so if you try to get RNA extracted from the inner parts of tissues then maybe you get some yield hopefully, but with compromised quality and in such case you must check your RNA integrity before subjecting to cDNA step and qPCR.
What we do normally that immediately after excision, dip some of the tissue in RNALater and you can get ideal results then. The rest of the chunk you can use for staining or preserve as FFPE blocks. Kindly check the following link for RNALater details;
https://www.thermofisher.com/pk/en/home/brands/product-brand/rnalater.html
On the contrast if you use FFPE samples and you have an optimized protocol with any best FFPE extraction kit (column-based or magnetic-beads based) then you are at right track. It depends what you want to use, but still FFPE extraction is cumbersome, lengthy, expensive and may or may not yield good results and a thorough optimization of your protocol is required. So the best is still to use fresh tissues for RNA extractions for the qPCRs and store your fresh tissue samples in RNALater, then you wont have any problem.
Regards....
Thankyou both for your response. We do normally used RNAlater but the same had come into us in formalin this time. I actually had both FFPE and formalin tissues from the sample sample. The target RNA is double stranded so not as labile as other RNA. For interest I had a Ct of 34 from the formalin sample and a Ct of 27 for the FFPE. So that indeed answered my question nicely!
thanks you again
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