Identification and validation of reference genes for RT-qPCR analysis in fetal rat pancreas

Abstract

The choice of reference gene is crucial for quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) assay. To screen and determine the suitable reference genes in fetal rat pancreas, we selected eight candidate reference genes (Gapdh, Actb, Rn18 s, B2m, Rpl13a, Tbp, Ywhaz and Ubc), and evaluated the constancy of gene expression from fetal rat pancreases in non-pathological situation and prenatal dexamethasone exposure (PDE) model, using four algorithms: GeNorm, NormFinder, Bestkeeper and Comparative ΔCt method. In addition, the alteration of mRNA levels of pancreatic insulin was compared between control and PDE groups to validate the reliability of selected reference genes for data normalization of RT-qPCR. The comprehensive ranking of reference genes under physiological condition was as follow: Gapdh >Actb >Ywhaz >Ubc >Rn18s >Rpl13a >B2m >Tbp (female); Actb >Ywhaz >Gapdh >Ubc >B2m >Rpl13a >Rn18 s | Tbp (male). The top ranking reference genes were also stably expressed in PDE fetal pancreas. The best reference gene combinations are: Ywhaz+Actb for female and Ywhaz+Gapdh for male fetal rat pancreas, respectively. Compared with low ranking or single reference gene, the change trend of insulin mRNA normalized by the best reference gene combination between control and PDE groups was more significant and consistent with that of serum insulin level. In conclusion, our results provided the optimal combination of stable reference genes for RT-qPCR assay in pancreatic developmental toxicity study.