Identification Of Suitable Endogenous Control MicroRNA Genes In Normal Pig Tissues

MicroRNAs (miRNAs) are a class of small (approximately 22-nucleotide), non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. They play key roles in a variety of biological processes, including development, cell proliferation and differentiation, apoptosis, and metabolism. For a better understanding of their roles in biological processes, q-PCR-based techniques are particularly attractive for miRNA profiling.In order to produce reliable q-PCR expression data, endogenous control (EC) genes are widely used to adjust for these variables which are not a result of the experimental design. Ideal EC genes are expected to be expressed at a constant level in the different tissues at all stages of development, should be unaffected by experimental treatments, and should pass through the same steps of analysis as the gene to be quantified. However, one must still validate the chosen endogenous control or set of controls for the target cell, tissue, or treatment, as no single control can serve as a universal endogenous control for all experimental conditions. Therefor, it is critical to evaluate and validate expression stability of EC genes in q-PCR expression.In this study, the expression stabilities of 13 selected porcine EC miRNA genes (5S,Met-rRNA,ssc-miR-16,ssc-miR-17,ssc-miR-23a, ssc-miR-24,ssc-miR-27a, ssc-miR-103,ssc-miR-106a,ssc-miR-107,ssc-miR-186,ssc-miR-221 andU6) were compared in all 47 tissue-specific normal tissues, ten types of adipose tissue, and four types of muscle tissue using an EvaGreen q-PCR approach. The stabilities of the 13 EC genes across three tissues groups were analyzed using geNorm algorithm which calculates the average expression stability values (M) for a candidate gene and expresses it as the average pairwise variation (V) for that gene with all other tested candidate genes. Our study showd that:(1) To analyze expression stability of EC genes, of the 13 EC genes,7 genes exhibited credible stability in all 47 surveyed tissues(M< 1.50), ssc-miR-17/ssc-miR-103 were the most stable gene pairwise (M=0.997).12 and 11 genes exhibited credible stability in the ten fat-type tissues group and four muscle-type tissues group respectively. ssc-miR-17/ssc-miR-107 exhibited the highest stability (M=0.374) in the ten fat-type tissues group and ssc-miR-17/ssc-miR-23a exhibited the highest stability (M=0.271) in the four muscle-type tissues group, while the U6 gene was the least stable gene in the three groups.(2) To analyze pairwise variation of EC genes, theoretically, we found 11 (V= 0.183),5(V=0.096), and 6 (V=0.110) EC genes in all 47 samples, ten fat-type and four muscle-type tissues groups with the lowest V values, respectively. In terms of economical and experimental feasibility, we recommend the use of the three most stable EC miRNA genes for calculating the normalization factor(NF), i.e., ssc-miR-17,-103 and-107 for all 47 different tissues. We also suggest that two sets of EC miRNA genes are appropriate for ten types of adipose tissue (ssc-miR-17,-107 and-24) and four types of muscle tissue (ssc-miR-17,-23a and-103), respectively.(3) Our analysis clearly showed that three optimal EC genes are adequate for an accurate normalization, which correlated well with the theoretical optimal number (r≥0.841,p<0.01).We envision that these results will serve as a valuable reference for other studies aimed at measuring tissue-specific miRNA abundance in porcine samples.