Hi dear researchers,
I am trying to amplify a region of the genome of a chlamydia-like organism from Formalin-Fixed,Paraffin-embedded (FFPE) tissue samples using a commercial kit for this purpose (QIAGEN). The chlamydial-like inclusions are checked using histological techniques, and after performing total DNA extractions, PCR bands are faint or not present.
I have performed end-point-PCR using fresh (not FFPE) tissue, obtaining good results, thus the PCR protocol is OK. So, my question is directed to obtain help in improving this kind of DNA extractions followed by end-point-PCR using FFPE tissue samples.
Best regards
Adrián Fuentes
Look for a better tool to remove paraffin, such as mineral old (Promega kit), or sth similar. Add an house-keeping gene PCR to check quality of DNA extraction.
Hi Adrian,
Something to consider is the length of your PCR target region. Formalin fixation can result in fragmentation of DNA, so the longer your amplicon the less likely you are to amplify it. This could explain why the PCR works well on the fresh material, where little fragmentation will be present.
Your options could be to re-design your PCR for a shorter amplicon, or try and find an extraction protocol that maximises the length of your DNA, there seems to be several papers on pubmed about the latter (eg http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2764035/ ).
Hope that helps,
Adam
dear Adrian Fuentes go through this article
High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil
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