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

Ruoyu Song; Shijun He; Yongbin Wu; Wanxin Chen; Jie Song; Yaxi Zhu; Huan Chen; Qianlu Wang; Shouman Wang; Sichuang Tan; Sipin Tan

doi:10.1016/j.heliyon.2023.e15269

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

Heliyon. 2023 Apr 7;9(4):e15269. doi: 10.1016/j.heliyon.2023.e15269. eCollection 2023 Apr.

Authors

Ruoyu Song^{1

2

3}, Shijun He^{1

2

3}, Yongbin Wu^{1

2

3}, Wanxin Chen⁴, Jie Song^{1

2

3}, Yaxi Zhu^{1

2

3}, Huan Chen^{1

2

3}, Qianlu Wang^{1

2

3}, Shouman Wang⁵, Sichuang Tan⁶, Sipin Tan¹

Affiliations

¹ Department of Pathophysiology, School of Basic Medicine Science, Central South University, Changsha, Hunan 410078, PR China.
² Sepsis Translational Medicine Key Laboratory of Hunan Province, Central South University, Changsha, Hunan 410078, PR China.
³ National Medicine Functional Experimental Teaching Center, Central South University, Changsha, Hunan 410078, PR China.
⁴ Department of Laboratory Medicine, The Second Xiangya Hospital, Central South University, Changsha, Hunan, 410011, PR China.
⁵ Department of Traditional Chinese Medicine, The Third Xiangya Hospital, Central South University, Changsha, Hunan, 410008, PR China.
⁶ Department of Thoracic Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, 410011, PR China.

Abstract

Objective: To screen and validate reference genes suitable for gene mRNA expression study in peripheral blood mononuclear cells (PBMCs) between septic patients and healthy controls (HC).

Methods: Total RNA in PBMCs was extracted and RT-qPCR was used to determine the mRNA expression profiles of 9 candidate genes, including ACTB, B2M, GAPDH, GUSB, HPRT1, PGK1, RPL13A, SDHA and YWHAZ. The genes expression stabilities were assessed by both geNorm and NormFinder software.

Results: YWHAZ was the most stable gene among the 9 candidate genes evaluated by both geNorm and NormFinder in mixed and sepsis groups. The most stable gene combination in mixed group analyzed by geNorm was the combination of GAPDH, PKG1 and YWHAZ, while that in sepsis group was the combination of ACTB, PKG1 and YWHAZ.

Conclusion: Our first systematic analysis of the reference genes in PBMC of septic patients suggested YWHAZ was the best candidate. The combination of ACTB, PKG1 and YWHAZ could improve RT-qPCR accuracy in septic patients. Our results identified the most stable reference genes to standardize RT-qPCR of sepsis patients, which can serve as a useful tool for gene function exploration in the future.

Keywords: RT-qPCR; Reference gene; Sepsis.