Validation of reference genes for cardiac RT-qPCR studies spanning the fetal to adult period

Many genes used as internal controls for mRNA expression studies are unstable (change) over development. This study determined an approach to validate reference genes for mRNA studies spanning the fetal period to adulthood in sheep hearts. We determined the mRNA expression of 12 candidate reference genes (ACTB, GAPDH, H3- 3A, HYAL2, PPIA, RNA18S1, RPL32, RPL37A, RPL41, RPLPO, RPS15, and YWHAZ) via RT- qPCR. Per RefFinder, which incorporates computational algorithms by BestKeeper, compar- ative delta Ct, GeNorm, and NormFinder, RPL32, RPL37A, HYAL2, ACTB and GAPDH were the most stable reference genes, although none were unchanged across all ages. Systematical calculation of the geometric means of 3 reference genes revealed the combina- tion of HYAL2, RPL32, and RPL37A was unchanged across the 5 fetal, neonatal, and adult ages. We determined the most stable combination of reference genes for cardiac gene expression studies in sheep from fetus to newborn to adult; these steps are applicable to determine inter- nal controls for mRNA studies in other organs, other species, and periods in which reference gene instability is high.