Selection and validation of optimal endogenous reference genes for analysis of quantitative PCR in four tissues pathologically associated with Kidney-yang deficiency syndrome following influenza A infection

Yepei Fu; Jia Yang; Shanshan Fan; Shaozhe Zhao; Ruikun Du; Syed Muhammad Ali Shah; Muhammad Akram; Rong Rong; Yong Yang

doi:10.3892/etm.2020.9374

Selection and validation of optimal endogenous reference genes for analysis of quantitative PCR in four tissues pathologically associated with Kidney-yang deficiency syndrome following influenza A infection

Exp Ther Med. 2020 Dec;20(6):244. doi: 10.3892/etm.2020.9374. Epub 2020 Oct 22.

Authors

Yepei Fu¹, Jia Yang¹, Shanshan Fan¹, Shaozhe Zhao¹, Ruikun Du¹, Syed Muhammad Ali Shah², Muhammad Akram², Rong Rong^{1

3}, Yong Yang^{3

4}

Affiliations

¹ College of Pharmaceutical Science, Shandong University of Traditional Chinese Medicine, Jinan, Shandong 250355, P.R. China.
² Department of Eastern Medicine, Directorate of Medical Sciences, Government College University of Faisalabad, Faisalabad, Punjab 38000, Pakistan.
³ Shandong Provincial Collaborative Innovation Center for Antiviral Traditional Chinese Medicine, Jinan, Shandong 250355, P.R. China.
⁴ Experimental Center, Shandong University of Traditional Chinese Medicine, Jinan, Shandong 250355, P.R. China.

Abstract

Kidney-yang deficiency syndrome (KYDS) infected with the influenza virus is a suitable model to imitate a population at high-risk to influenza infection with a high rate of morbidity and mortality. However, the specific molecular mechanisms underlying this disease remain unclear. A stable reference gene is essential as an internal control for molecular biology research of this condition. Reverse-transcription-quantitative PCR (RT-qPCR) is considered an extremely sensitive technique used for absolute and relative quantification of target genes transcript levels. To accurately estimate the relative expression of genes in cells from mice with KYDS in response to infection with influenza A virus subtype H1N1 (A/H1N1) virus using RT-qPCR, it is necessary to identify suitable reference genes. In the present study, analysis of 10 reference genes (Act-β, β2m, GAPDH, Gusβ, Tubα, Grcc10, Eif4h, Rnf187, Nedd8 and Ywhae) was performed across a set of 4 tissue types: Lung; heart; liver; and kidney. KYDS mice were inoculated with A/H1N1 virus or a mock control. For analysis, geNorm, BestKeeper, NormFinder, and Bio-Rad Maestro^™ statistical programs were used for the estimation of the stability of the reference genes. The results were authenticated through extended experimental settings using a group of 10 samples, parallel to 3 additional innate immune system-associated genes of the host, TLR3, TLR7 and RIG-I, which were also analyzed using the same algorithms. From the 4 algorithms, taking into account the joint analyses of the ranking order outputs, the 2 genes Ywhae and Nedd8 were identified to be the most stable for mice with KYDS following infection with A/H1N1 virus. In contrast, the least stable genes in all 4 tissues were GAPDH and β2m. These results may affect the choice of reference genes in future studies that use RT-qPCR analysis of target genes in experimental conditions, such as mice with KYDS infected with influenza A virus.

Keywords: Kidney-yang deficiency syndrome; influenza A virus; influenza A virus subtype H1N1; reference genes; reverse transcription-quantitative PCR.