Identification of stable endogenous reference genes for real-time PCR in the human fetal gonad using an external standard technique.

Measurement of tissue mRNA transcript levels is critically dependent upon the normalization strategy used. For real-time polymerase chain reaction (PCR) (RT-PCR) this commonly depends upon identification of stable (non-variable) endogenous reference genes, with housekeeping genes (HKGs) the most commonly used. In this report we describe the use of an external mRNA standard to identify stable HKGs in the human fetal gonad. Total RNA was extracted from second trimester human fetal gonads and a standard amount of luciferase mRNA was added at the start of the extraction process. Levels of luciferase were then measured relative to each of seven HKGs (SDHA, TBP, B2M, PMM1, SFRS4, HMBS and UBC) by RT-PCR. When normalized to tissue weight, HKG expression was constant across fetal ages. Measurement of overall variation in transcript expression showed that PMM1 was the most stable HKG in the ovary while B2M was most stable in the testis. Re-analysis of the data using GeNorm and NormFinder algorithms showed that two of the top three most stable HKGs were the same using all three methods. This study describes a method for identification of endogenous, stable reference genes for RT-PCR studies of transcript expression levels which is objective and not dependent on prior assumptions of HKG expression. This technique is likely to be applicable to most tissues and, in this case, identifies suitable HKGs for studies into human gonadal development.