Our group experiences problems with poor DNA extraction quality from FFPE material. FFPE material from other institutions pose no problems. Influence of different formalin fixation has been ruled out. Are there other common factors which can result in these problems? Thank you!
Have you checked if your formalin is buffered?. Which type of samples are you trying to obtain DNA?, and finally how old are those samples?
Javi
Have you tried with ¨Infinium FFPE DNA Restoration Solution¨ by Illumina? looks like it can repair these degraded DNA samples in preparation for use with the Infinium HD Assay. Good luck!
What do you mean by "poor extraction quality"? How have you ruled out the formalin issue? What method of DNA extraction do you use?
What tissue are you working with?
Hi David,
I am also curious about the method you are using. Based on the information you have provided, what I would like to do is offer you a free sample of the method ( kit) we use here at MO BIO. We have a kit to extract DNA from FFPE samples and we just release a kit to extract RNA. I would be happy to send you a sample of each of them if you want to try. http://www.mobio.com/tissue-cells-dna-isolation/biostic-ffpe-tissue-dna-isolation-kit.html
Let me know
Thank you all! The Formalin Issue was ruled out by trying to extract DNA from FFPE material from different archive years, some were fixed using buffered formalin, some were fixed using unbuffered formalin. The amount of extracted DNA is poor. Compared with blocks from other institutions, we can only extract around 10% the amount of DNA when using our own blocks. The tissue is human breast carcinomas. I'll have to ask what the exact DNA extraction method is, since I am not doing this myself, but we haven't found the problem yet.
Hi David,
to be able to help you, it is important that you answer the questions asked you:
What method of DNA extraction do you use?
Are you using a specific kit? If the answer is yes, what kit is it?
What tissue are you working with?
How old are those samples?
Could you give more details about your method of extraction? Do you use kits or follow the chloroform protocol? What kind of tissue you are working on? How do you measure your DNA concentration? How is the curve?
Many salts and reagents may cause poor quality and bizarre curves. In such cases, more washes may work. Sometimes, you may have some undigested floating stuff at the end with unknown origin (rarely happens, but happens!), you can spin the final yield and recover the supernatant. Anyway, all these solutions depend on the answer of the above questions.
As a rule of thumb, Kits usually give you less but cleaner yield. However, you may face poor quality issues with kits as well.
Hi David,
try to ask to the other institution what type of kit or methods they used for the extraction in the order to know if it is the same or not
Dear Simone, the DNA is extracted centrally so the extraction method is always the same (I'll try to find out what method). The extraction is not done at our institution. What differs are the FFPE blocks and there is only a problem with our FFPE blocks. FFPE blocks from other institutions yield more DNA. We know that a few years ago at our institution unbuffered formalin was used. But the problem also occurs with FFPE blocks from the years when buffered formalin was used. But there obviously seems to be a problem with the fixation at our institution. The FFPE material is one to several years old. Regards, David.
Dear David,
if the problem is your blocks, while those of the other institutions give good yields of DNA, then you could ask the other institutions as they prepare their blocks and compare it with your method
DNA extraction is a laborious job and need much care in the selection of extraction method. chemicals used in extraction should be working ones. so u should use fresh chemicals and try some other method and also compare the results of various methods. i hope u will too get good quality and quantity of DNA. good luck
Dear David,
Are the other blocks you are comparing with also breast samples? Because the yields from breast samples are generally lower due to the high amount of fat tissues. How much DNA are you managing to extract?
You should clarify if you are using blocks vs slides. Also, the age is an important factor. Then, you will find many here to help you.
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