Basic & Clinical Medicine ›› 2021, Vol. 41 ›› Issue (1): 87-92.

• Original Articles • Previous Articles     Next Articles

Validation of reference genes for the normalization of the RT-qPCR in peripheral blood mononuclear cells of patients with Takayasu arteritis

TIAN Yi-xiao, LI Jing*   

  1. Department of Rheumatology and Immunology, Key Laboratory of Rheumatology and Clinical Immunology, Ministry of Education, National Clinical Research Center for Dermatologic and Immunologic Diseases (NCRC-DID), Peking Union Medical College Hospital, CAMS & PUMC, Beijing 100032, China
  • Received:2019-11-18 Revised:2020-04-30 Online:2021-01-05 Published:2020-12-30
  • Contact: *

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.

Key words: Takayasu arteritis, real-time polymerase chain reaction, RNA stability, selection of reference gene

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