For our study, I need to extract DNA from FFPE mammary tissue. Unfortunately, some samples do not lyse well when using the QiaAmp FFPE DNA extraction kit. Has anyone had the same experience and possibly found a solution?
And what can be the cause?
Can fatty tissue hinder the lysis process?
Hopefully, someone can help me to tackle this problem.
Thanks in advance!
Perhaps you have problems with aged samples. Try fresh proteinase K stored at the correct temprature. take into consideration that residual formalin could nhibit proteinase K.
Thanks you very much for your answer.
Unfortunately, the problem lies with the most recent samples. I have the impression that older samples lyse much quicker then recent samples.
The proteinase K is stored at the right temperature and the lyses buffer does not contain any precipitant.
If anyone else has this same challenge, please let us know how you have won it!
The moment I find a solution (other than increasing lysis buffer and proteinase K) I will report it.
Thanks in advance for your help.
Thanks for your answer. May I ask how you hiomogenized the tissue?
You can try to 'crusch' the samples more by pressing with Pestles (Kimble Kontes) in de tip of 1.5 mL eppendorf tubes.
Proteinase K incubation at 56 degrees for about 16-18 hours when applied on tissue (agitate the tubes for proper mixing).
Does your buffer/digestion mix also contain Iodoacetamine and Pepstatin A?
You could cut finely the tissue using a sterile scalpel to ensure complete digestion. There is another method, which involves grinding with liquid nitrogen, but literature has shown that this method can cause DNA degradation.
Then you could continue by using a lysis buffer to break down cell membrane, sodium dodecyl sulphate which also helps in break down of cell membrane and Proteinase K.
This should be incubated overnight at the appropriate temperation (approx 50 degrees)
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