Jichu yixue yu linchuang (Jan 2021)
Validation of reference genes for the normalization of the RT-qPCR in peripheral blood mononuclear cells of patients with Takayasu arteritis
Abstract
Objective To validate proper reference genes for quantitative real-time polymerase chain reaction (RT-qPCR) used for comparing mRNA expression levels in Takayasu arteritis' (TAK) and healthy controls' (HC) peripheral blood mononuclear cells (PBMC). Methods Total RNA in PBMCs was extracted and used RT-qPCR to determine the profiles of 9 candidate genes, including β-glucuronidase, GAPDH, ACTB, SDHA, HPRT1, RPL13A, B2M, YWHAZ and PKG1. Then compared their transcription stability by geNorm, NormFinder, and BestKeeper. Afterwards, with T-bet, GATA3 and RORC as the targeted genes,explored the influence of reference genes with different stability on mRNA relative abundance. Results The gene combination of B2M-SDHA was selected by geNorm, and HPRT1 was the most stable one in analysis results of NormFinder and BestKeeper, while GAPDH was less stable. Conclusions Genes that have been expressed stably may be upregulated or downregulated when patients with autoimmune diseases received immunosuppressive drugs. When the sample size is small, the more stable internal reference may facilitate the identification of inter-groups difference.
