Housekeeping gene normalization in real-time PCR is it always necessary?

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Soner Öner-Sieben

Hi Moritz,

of course you can use two HKGs without validation which would be more accurate than using just one HKG. However, as long as you don't empirically test a panel of HKGs and validate them you can't be sure whether your HKGs might be differentially expressed. Hence, your relative expression values for your GOI would be comprimised.

Best regards

Rheza Andika

It's essential that the housekeeping gene should not vary in the tissues or cells under investigation, or in response to experimental treatment. Since you use various cancer cell lines, I think you should choose the most stable housekeeping gene for those cells. The reference you used, Vandesompele, et al. (2002), have measured and ranked some control genes (included GAPDH dan beta actin) based on their stability on different tissues.

If you want to use two housekeeping genes, still, you better choose the the genes with the expression stability consideration.

Moritz Ponert

Thank you very much for your help!!!!