Hello everyone,
I am currently working with cancer tissues fixed in FFPE blocks, but despite numerous attempts, I am unable to obtain an amplicon of my gene of interest. Here are the details of my process:
The PCR conditions were optimized using DNA extracted from fresh blood and DNA from tissues prepared with home-made extraction solutions, both of which yielded successful amplifications.
Given these efforts, what additional steps or modifications would you recommend to achieve successful amplification from my FFPE samples ?
Thank you for your help!
you may not be getting very much dna or it may be highly fragmented from your ffpe section. Nested pcr may help in getting a strong positive signal
Thank you very much for your response. Indeed, we are obtaining highly fragmented DNA as evidenced by the agarose gel, despite the substantial concentration reported by the Nanodrop.
Attached, you will find images of the agarose gels from our DNA extractions: one from tissues and another from fresh blood, where amplification was successful and the amplicon size is 240 bp. Also included are the current PCR results obtained after using the FFPE extraction kit.
The nested PCR approach is a valuable suggestion, and we will certainly consider it. Thank you again for your assistance.
do not worry about nanodrop readings. It measures all nucleic acids however badly degraded and even dNTPs. It measures amount not quality and in fixed specimens you expect a lot of degradation
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