I am working with FFPE blocks from cancer and trying to
figure out relative amount of a specific gene expression in cancer samples.
We have RNeasy FFPE kit to extract RNA and test qRT-PCR.
We also could use in situ hybridization for the same gene (RNAscope).
I have worked with RNA in cell lines but am skeptical about
extracting RNA from 10-years old FFPE tissues for quantification.
Can anyone give some advice on which method I should try out first?
- The FFPE will actually preserve your RNA so with specilist kits, designed to extract RNA from such samples then qRT PCR will be where I start
- I would however take a look at individual messages by RNAScope especially if you are extracting RNA with standard kits:
- There are speciliast kits that are likely to recover RNA in more abundance and with represnetative molar equivalents in your recovered RNA population
Laurence Stuart Dawkins-Hall Thank you for the advice. I will try both methods and also investigate in kits that may help my research.
The FFPE will potentially cross-link and degrade your RNA so I would do some careful QC after RNeasy extraction to be certain that samples to be compared to each other (e.g. normal adjacent vs tumor) have similar QC profiles and will not introduce PCR bias. Droplet digital PCR would be the ideal way to quantitate---even better if you have a target to standardize against that is unaffected in tumors and controls. You could assay these simultaneously with different dyes.
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