Hi All
I am extracting RNA from paraffin blocks from liver colorectal mets and normal liver for PCR, when i did the PCR the b actin and GAPDH genes are not expressed.
I think the reasons may be low quality RNA (RIN about 2.5 in all of samples) or I need to try different housekeeping genes?
Any ideas?? would be much appreciated on how I can overcome this problem
Many thanks
Eirini
Hi,
I guess the quality of your RNA plays a significant role here. Other parameters could be
- Used strategy for RT-Priming: Eould propose to use handom hexamer plus Oligo DT
- Length of you amplicon: shorter amplicons have a higher amplification tendency in the case of fragmented RNA
- Does you PCR per se work optimal?
- How are you extracting the RNA? Is there perhaps another option that improves RNA quality?
In general FFPE is not an easy starting mateiral but there are solutions to tackle your problems
Sven
Thank you very much!
Yes I agree the quality of RNA quality is the issue. PCR per se works well as I have done samples for cell lines with no issues
I use the norgen kit (xylene, ethanol, proteinase k, to extract RNA)
I will discuss your suggestions with my supervisor
thank you very much again
BW
Eirini
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