Suitable internal control microRNA genes for measuring miRNA abundance in pig milk during different lactation periods

Determination of an optimal set/number of internal control microRNA (miRNA) genes is a critical, but often undervalued, detail of quantitative gene expression analysis. No validated internal genes for miRNA quantitative PCR (q-PCR) in pig milk were available. We compared the expression stability of six porcine internal control miRNA genes in pig milk from different lactation periods (1 h, 3 days, 7 days, 14 days, 21 days, and 28 days postpartum), using an EvaGreen q-PCR approach. We found that using the three most stable internal control genes to calculate the normalization factor is sufficient for producing reliable q-PCR expression data. We also found that miRNAs are superior to ribosomal RNA (rRNA) and snRNA, which are commonly used as internal controls for normalizing miRNA q-PCR data. In terms of economic and experimental feasibility, we recommend the use of the three most stable internal control miRNA genes (miR-17, -107 and -103) for calculating the normalization factors for pig milk samples from different lactation periods. These results can be applied to future studies aimed at measuring miRNA abundance in porcine milk.