Abstract: 【Objective】Reference gene is the key to obtain the accurate analysis results when apply quantitative RT-PCR (qRT-PCR) technique to identify the gene expression level. However, reference gene and microRNA (miRNA) which are used in researching the molecular biology of porcine testis are not well characterized. Screening suitable reference gene will provide a reliable evidence for the quantitative analysis of target gene in different periods of porcine testis tissues.【Method】Porcine testis tissues at 8 different developmental stages (E 90, D 1, D 30, D 60, D 90, D 120, D 150, and DM) were selected as materials in this study. Trizol method was used to extract the total RNA and the concentration and purity of total RNA were detected by ND-2000 NanDrop spectrophotometer. The primers of 5 commonly used protein coding reference genes (GAPDH, TBP, β-actin, SDHA and B2M) and 5 miRNA reference genes (U6, ssc-miR-17-5p, ssc-miR-26a, ssc-miR-27a and ssc-miR-103) were designed and synthesized to reverse transcription. cDNA templates were diluted to 7 concentration gradients with 1:10 serial dilution to construct standard curves. The expression of 10 candidate reference genes in porcine testis tissues was systematically analyzed at specified times, then the Genorm 3.5 was used to analyze the results. The most stable reference genes were picked up according to the stability value which called M value, the smaller the value of M, the better the stability of the reference gene; otherwise, the stability would be worse.【Result】The melting curves of 10 candidate reference genes (GAPDH, TBP,β-actin, SDHA, B2M, U6, ssc-miR-17-5p, ssc-miR-26a, ssc-miR-27a, and ssc-miR-103) indicated that the expression of all the candidate reference genes was of some specificity, no primer-dimers and nonspecific amplification. The relationship between Ct value and the logarithm of relative copy number was coincidence with linear relation according to the standard curves which were made with Ct value as ordinate and the logarithm of relative copy number as abscissas in a series of diluted concentration gradients. Analysis showed that the most stable reference gene of protein encoding gene was TBP and the most unstable was GAPDH. U6 was the most suitable gene for miRNA expression analysis and ssc-miR-26a was the most unsuitable. 【Conclusion】 This study has screened the most suitable reference genes (TBP and U6) to identify porcine testis gene expression level successfully. The highly ranked reference genes identified from this study can provide a theoretical basis for the selection of the most suitable reference gene to detect differences in expression rates of genes and miRNAs in porcine testis tissues.

Key words: porcine testis tissue, reference genes, qRT-PCR, miRNA