Hprt Serves as an Ideal Reference Gene for qRT-PCR Normalization in Rat DRG Neurons

To identify suitable reference genes for gene expression studies in rat dorsal root ganglia (DRG) neurons.

The raw cycle threshold (Ct) values of 12 selected reference genes were obtained via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in neurons at different developmental stages or under different treatments. Two strategies were employed to screen the most stable reference genes: the genes were ranked according to the coefficient of biological variation and further validated using geNorm and NormFinder programs. The stable and unstable reference genes were subsequently used as internal controls to assess their effects on target gene expression.

All reference genes showed varying degrees of fluctuation in Ct values during the growth process of neurons or after different treatments. 18S ribosomal RNA (Rn18s) and β-actin (Actb) exhibited the most significant changes, while ubiquitin C (Ubc), hypoxanthine phosphoribosyl transferase (Hprt), and mitochondrial ribosomal protein L10 (Mrpl10) showed relatively minor changes. The most stable and unstable genes obtained by different evaluation methods varied slightly. Overall, Actb was found to be the most unstable reference gene, while Hprt was the relatively most stable reference gene. The use of unstable reference genes Actb and ankyrin repeat domain 27 (Ankrd27) as internal controls led to high variability within the control group, ultimately affecting the determination of target gene expression. In contrast, the stable reference gene Hprt had small inter-assay variation and high stability.

Our observations indicate that Hprt is a proper endogenous reference gene for qRT-PCR analysis in rat DRG neurons and thus provides a critical molecular basis for the genetic characterization in neurological disorders.