Verification of Preferred Reference Genes for RT-qPCR Analysis of Radiosensitivity Gene in Human Peripheral Blood

High-throughput gene expression analysis represents a promising approach for evaluating irradiation doses in vitro. This study aimed to identify preferred reference genes for normalizing RT-qPCR data in human peripheral blood following X-ray irradiation.

We assessed the stability of GAPDH, 18S rRNA, UBC, HPRT, DPM1, ITFG1, MRPS5, and ACTB, which are commonly utilized in radiation biodosimetry studies. Three whole blood samples from healthy donors were divided into three culture time groups (2, 12, and 24 hours) and subjected to X-ray doses of 0, 0.5, 1.2, and 3.5 Gy, resulting in a total of 12 subgroups. NormFinder, geNorm, BestKeeper, and ΔCt were employed to evaluate the stability of the candidate genes. RefFinder was utilized to comprehensively rank the candidates and identify suitable reference genes.

The preferred reference genes identified were UBC, HPRT, and GAPDH for 2-hour culture time; for 12 hours, UBC, HPRT, and 18S rRNA were preferred; and for 24 hours, 18S rRNA, MRPS5, and GAPDH were preferred.

This study presents novel findings of reference genes suitable for normalizing RT-qPCR data following X-ray irradiation in human peripheral blood across three distinct culture periods. It offers valuable insights for the selection of reference genes in radiation biodosimetry research.