Reference gene is unstable — normalization fails (qPCR (RT-qPCR))

Question

Accepted Answer

Reference gene Ct varies > 1 cycle between samples; trend of reference vs target gene disagrees; normalization gives strange results. Common causes: 1) Reference gene is affected by the experimental treatment (no longer "housekeeping" under this condition) 2) Reference gene varies between sample types / tissues 3) Wrong choice of reference gene for this biological context 4) Reference primer amplification efficiency differs from target Solutions: 1) Test multiple candidate reference genes (GAPDH, β-actin, 18S, HPRT1, RPL13A) and pick the stable one(s) 2) Use the geometric mean of 2 – 3 reference genes 3) Validate stability with geNorm / NormFinder / BestKeeper software 4) Confirm primer efficiency (90 – 110 %, R² > 0.99) for both target and reference