I want to isolate RNA in high quality to be used in RNAseq experiment from Paraffin or formalin. What i want to know are:
1- The optimum temperature for saving tissues in formalin or paraffin
2- The time of preservation to get high quality RNA
3- Which one is the best one.
Length of time in formalin is the concern: the longer the time in formalin the more cross-linking of RNA to protein and the more adducts and methylol bridging of various ribonucleotides as well on account of HCHO action (48 hours is generally long enough in formalin). Also, loss of mRNA poly A tails can be expected if over-fixed. Paraffin embedding usually happens after formalin fixation.
Stopping tissue RNAses upon tissue collection is your main concern: So, why not just homogenize your tissues in TRIzol right upon tissue collection (e.g., 0.3 g of tissue homogenized in 3 mL cold TRIzol) then store the slurries at -80C until isolating the total RNA using the standard TRIZol approach. You can store your TRIzol tissue homogenates at -80C for a long time - and isolate RNA from them at your leisure. I always get RNA of high quality this way: Bioanalyzer RIN values of 8.0 or greater.
I would concur with Jack. If what you want is high quality RNA, you shouldn't fix in formalin at all, but freeze the tissue or process it right after collection.
Formalin fixation followed by paraffin embedding is not considered usually as an ideal source for isolating RNA, however it is quite possible if the proteins are properly retrieved. As we all know the penetration rates for formaline in tissue is around 2mm/hour, longer the tissue remains in formaline would have some deleterious effects isolation of RNA. In addition thickness of tissue is also important, is the sample is less than 2 mm in thickness, formaline might be penetrating earlier. If by any means formaline and/or paraffin are inevitable to be used, the whole protocol can be sent, provided the sample size, nature, thickness and other parameters are known. Frozen sample is readily available but storage is difficult and expensive. Formaline fixed paraffin embedded tissues can be stored even at room temperature.
Like everybody is saying, formalin-fixed tissue is not an ideal source for isolating RNA. Though there is protocol which you can use the formalin-fixed tissue as a normalisation parameter for later on RNA extraction on your fresh frozen tissue, I think it's time consuming and costly when you have a choice on how to process your tissues at the first place.
We store the tissue in RNAlater immediately after collection. Long term in -20C and short-term in 4C fridge. Haven't said that it is always good to not have the tissue to store for an extended period, process the tissue asap would be ideal.
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