Determination of Reference microRNAs for Relative Quantification in Porcine Tissues

Abstract

Relative quantification is the strategy of choice for processing RT-qPCR data in microRNAs (miRNAs) expression studies. Normalisation of relative quantification data is performed by using reference genes. In livestock species, such as pigs, the determination of reference miRNAs and the optimal number of them has not been widely studied. In this study, the stability of ten miRNAs (Ssc-let-7a, Ssc-miR-103, Ssc-miR-17-3p, Hsa-miR-25, Hsa-miR-93, Ssc-miR-106a, Ssc-miR-191, Ssc-miR-16, Ssc-miR-26a and Ssc-miR-17-5p) was investigated by RT-qPCR in different tissues (skeletal muscle, kidney, liver, ovary and uterus) and in different pig breeds (Iberian, Landrace, Large White, Meishan and Vietnamese) as variation factors. Stability values were calculated with geNorm and NormFinder algorithms obtaining high correlation between them (r2 = 0.99). The analyses showed that tissue is an important variability factor in miRNAs expression stability whereas breed is not a determinant factor. All ten miRNAs analysed had good stability values and, therefore, can be used as reference miRNAs. When all tissues were considered, miR-93 was the most stable miRNA. Dividing data set by tissues, let-7a was the most stable in skeletal muscle and ovary, miR-17-5p in kidney, miR-26a in liver and miR-103 in uterus. Moreover, the optimal number of reference miRNAs to be used for proper normalisation data was determined. It is suggested the use of five reference miRNAs (miR-93, miR-25, miR-106a, miR-17-5p and miR-26a) in multi-tissue experimental designs and the use of three reference miRNAs as the optimal number in single tissues studies (let-7a, miR-17-5p and miR-25 in skeletal muscle; miR-17-5p, miR-93 and miR-26a in kidney, miR-26a, miR-103 and let-7a in liver, let-7a, miR-25 and miR-106a in ovary and miR-103, let-7a and miR-93 in uterus). Overall, this study provides valuable information about the porcine reference miRNAs that can be used in order to perform a proper normalisation when relative quantification by RT-qPCR studies is undertaken.

Addition of coconut oil to the diet based on maize dried distilled grains with solubles (DDGS) alters miR-122a expression in the pig liver

“…The obtained results on the stability of miRNA expression are in partial agreement with those of Timoneda et al (2012) who investigated the stability of ten miRNAs in various tissues and breeds of pig. It was observed that all investigated miRNAs were stable and the expression of selected miRNAs was Figure 2.…”

“…Furthermore, the miR-335 was included in the study despite the fact that in the previous study (Pawlina et al, 2015) we did not detect it in the liver miRNAome by NGS (Next Generation Sequencing) but as many as seven DEG were possible targets for this miRNA. The let-7a was also included in the experiment because it has been shown to be a good endogenous control in the porcine liver (Timoneda et al, 2012).…”

miRNA Expression Profile Analysis in Kidney of Different Porcine Breeds

“…According to sequence count, nine miRNAs highly expressed (Hsa-miR-200b-3p, Hsa-miR-200c-3p, Ssc-miR-126, Ssc-miR-126*, Ssc-miR-99a, Ssc-miR-532-5p, Ssc-miR-92a, Ssc-miR-26a and Bta-miR-193b, n>100) and four miRNAs lowly expressed (Ssc-miR-423-5p, Ssc-miR-29c, Ssc-miR-486 and Ssc-let-7f, n<100) in the kidney miRNAome were selected to measure their expression levels by RT-qPCR. Hsa-let-7a, Hsa-miR-25 and Hsa-miR-93 were used as reference miRNAs based on literature [50], [51]. Primers used were designed by Exiqon® (Denmark).…”

Feeding of Enterococcus faecium NCIMB 10415 Leads to Intestinal miRNA-423-5p-Induced Regulation of Immune-Relevant Genes

“…The amplification program comprised an initial step at 95°C for 10 min, followed by an annealing step and an elongation step for 1 min at 60°C. Porcine miR-17, miR-103, and miR-17 served as reference miRNAs (25).…”