I am doing qPCR for osteogenic, adipogenic and chondrogenic genes using human MSC samples. I am using beta-actin as my reference gene and undifferentiated MSCs as my calibrator sample. When I normalize the data to the reference gene and calibrator sample, I get very high expression folds, sometimes up to 50 (using Pfaffl's method).
I am unable to figure out what is wrong as the expression of these genes should not be that high compared to the calibrator. There does not seem to be anything wrong with my qPCR as each melting curve shows only one peak, my NTCs show negative amplification (or very late Ct values), and the Ct values of my samples are between 15-30.
Before doing qPCR, I did RT-PCR and ran the samples on agarose gel. The band sizes were as expected.
What could be the possible causes for the abnormal increase in expression?