Hi everyone, I isolated RNA from FFPE PCa samples with very acceptable quality. CT values are very consistent. No more than 0.5 CT difference and all under 34. Now, i am selecting the best housekeeping control genes. For prostate cancer samples, what are the best control genes for normalization?
Ages ago I have looked for something similar for breast cancer, and the best approach is data-driven: look at an expression profiling study of a large cohort and instead of looking for the most variable genes (which is where everyone looks), look at the most stably-expressed ones (I usually look for small interquartile range or 95%-5% range). Pick 2-3 genes of varying abundance and preferably with different type of function (structural, metabolic, cell cycle etc) and use the geometrical mean of 2-3 as your reference.
From experience, generic "housekeeping" genes, such as GAPDH, Actin etc are actually more variable than you would guess, often in association with clinical parameters! In general, mRNAs encoding ribosomal proteins (e.g. rpL13a) tend to be very stable as they are predominantly regulated at the post-transcriptional level.
For breast cancer, by far the best (and now validated many times over in primary material, FFPE, cell lines) is eEF1A1. If it turns out to be suitable for you, I am happy to email you tried and tested primer sequences.
Good luck!
- Badges
- Science method