I have tried isolation of DNA from FFPE tissue using a variety of methods inclding qiagen kits and manual ctab method, with and without using Xylene. etc. I am trying to amplify a few cancer genes for my study. However most of the time one particular set of primer which gives a intact band in control whole genomic DNA isolated from blood fails to amplify in FFPE DNA while most of the my other primers give amplification in FFPE DNA.
I would like to understand what could be the reason behind this? Is the primer binding site more prone to random degradation?..
Suggestion please..
Hi Abhinav,
I also work with FFPE samples and gene expression analysis. Taking into account the amplicon size is very important. Everytime is possible I try to design primers shorter than 100bp. Another issue is that Ct values for FFPE samples usually come 5 cycles after than fresh isolates.
Best,
Ana
Hello Abhinav,
I certainly agree with the importance of your amplicon size.
If you want to verify the size of your DNA fragments, either run an agarose gel at low percentage (0.8%) or if available you can run the agilent bioanalyzer with the Agilent DNA 1000 Kit.
Usually fragments are around 150bp so your amplicon size must be designed accordingly.
Since you are using the qiagen FFPE kit, make sure to do the step at 90°C after the proteinase K, in order to remove secondary conformation that occur during FFPE, and which might affect you PCR efficiency.
Good luck,
Thanks for the suggestion..my amiplicon is around 300 bp.. I am intrested in sanger sequencing of my amplicon.. so i can not go below 150... i will try to dedesgn my primers with 150 bp amplicon.
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