I am starting a project to amplify DNA from FFPE (formalin-fixed parafin-embedded) tissue samples, and I have noticed that even when I spike the samples DNA from other sources, my amplification is not as good in those samples, even the DNA from the spike is not amplifying well. I am wondering if anybody has some advice about how to work with these samples, or ideas about how to counteract the formaldehyde remnants in my PCR.
Thanks!
Formaldehyde exerts mutagenic and carcinogenic effects by both damaging DNA and inhibiting DNA repair.Moreover it also causes endogenous cross linking between DNA and proteins and between single-strand breaks in DNA.
What I would suggest you if possible at least once extract the DNA right from the beginning all by yourself just to check whether there is any handling error or any type of processing error in the extraction step.After that proceed for a Gel Run to check the purity and integrity of the DNA ,simultaneously do a DNA Quantification just to check the initial DNA Concentration and note it down.
Now if it comes good enough to proceed for the next step which is your own experimental part you can either design primer that can amplify even a
This paper may help you: http://gump.auburn.edu/halanych/lab/Pub.pdfs/Schander&Halanych2003.pdf
This manuscript may also help. What size is your product? Cross-linking and subsequent degradation of DNA will always be a problem with formaldehyde. I can only sugget designing primers that will amplify as small a product as possible.
I worked with samples fixed with formaldehyde. If your target PCR product includes one copy gene or product longer than few hundred bp, it could be really risky work...
I don`t know if this will be practical but it could help to perform a first inactivation of formaldehyde with a sterile solution of histidine 20 g/l+glycine 5g/l during 30 min (minimum), however this contact time will depend on the sample thickness. After that I will try to extract the DNA with phenol:chlorophorm and then with chlorophorm follow with isopropanol precipitation and ethanol wash.
Do you really need to fix your sample with formaldehyde? Maybe try a precipitative fixation instead of a cross-linking on... the result will be better. Maybe embedding in OCT will be a good idea , too. The fixation and embedding is a lot faster and does not give the change of degradation (so much). Or you can try the microwave oven technique for embedding in paraffin. Embedding can be done in around 5 hours. For DNA Isolation of FFPE sections is also the Proteinase K digest step really important.
I've had decent luck getting DNA from frozen sections of formaldehyde fixed tissue for genotyping, I take a bit of tissue (a few scraped off sections, or the "stub" of a tissue block) and digest overnight in 400 ul of SNET + Proteinase K (4-5 ul of NEB stock works well). Digest at 60C overnight; this will help the proteinase K and the heat will help un-crosslink the DNA. Do a quick phenol-chloroform extraction and ethanol precipitation the next day to get the sample ready to use.
No matter what you do, the quality will be lower, but it should be sufficient for genotyping or other simple assays.
Thanks for the advice. The samples I am working with right now are from tumor tissue that was fixed with formaldehyde, and the DNA was extracted by another lab. We just get the DNA. I am trying to amplify the biggest product possible. What I am having trouble with right now is the PCR reaction itself. When I added extra DNA as a spike, I still did not get very good amplification compared to another sample with DNA from tissue that was not fixed with formaldehyde, spiked with the exact same amount of DNA, and amplified in the same reaction. Could there be something still in the sample that would be inhibiting all PCR?
The simplest way is to try re-PCR if you need to increase amount of amplicons.
Hi Elizabeth
What size is the PCR amplicon? As for inhibitors being carried over from DNA extraction, yes that can happen. Do you know the method they used to perform the extraction? Did you spec the DNA yourself? What is the 260/230 ratio? This measures contaminants like phenol, carbohydrates, etc... that can inhibit PCR. A good sample will have 260/230 better than 2.0.
Formaldehyde exerts mutagenic and carcinogenic effects by both damaging DNA and inhibiting DNA repair.Moreover it also causes endogenous cross linking between DNA and proteins and between single-strand breaks in DNA.
What I would suggest you if possible at least once extract the DNA right from the beginning all by yourself just to check whether there is any handling error or any type of processing error in the extraction step.After that proceed for a Gel Run to check the purity and integrity of the DNA ,simultaneously do a DNA Quantification just to check the initial DNA Concentration and note it down.
Now if it comes good enough to proceed for the next step which is your own experimental part you can either design primer that can amplify even a
Any luck with your pcr? I am looking at formaldehyde fixed samples (not embedded) and would be interested in your experiences. Regarding the actual pcr step I cam across this paper (http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0077771#pone-0077771-g008) that suggests that the inhibition is from small DNA fragments and suggests ways to overcome it.
This Article Optimization of DNA isolation method from Formalin-Fixed-Par...
suggests that it's difficult to obtain amplicons >450bp from fixed gDNA. Along this otherArticle Improved PCR Performance Using Template DNA from Formalin-Fi...
, there is evidence that fixation chops gDNA down to predominantly 500-bp, and that PCR is inhibited with gDNA inputs of ~1µg. This inhibition may be alleviated by increasing annealing and extension times, and increasing polymerase and dNTP amounts.- Badges
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