Discovery of porcine microRNAs and profiling from skeletal muscle tissues during development

Abstract

MiRNAs (microRNAs) play critical roles in many important biological processes such as growth and development in mammals. In this study, we identified hundreds of porcine miRNA candidates through in silico prediction and analyzed their expression in developing skeletal muscle using microarray. Microarray screening using RNA samples prepared from a 33-day whole embryo and an extra embryo membrane validated 296 of the predicted candidates. Comparative expression profiling across samples of longissimus muscle collected from 33-day and 65-day post-gestation fetuses, as well as adult pigs, identified 140 differentially expressed miRNAs amongst the age groups investigated. The differentially expressed miRNAs showed seven distinctive types of expression patterns, suggesting possible involvement in certain biological processes. Five of the differentially expressed miRNAs were validated using real-time PCR. In silico analysis of the miRNA-mRNA interaction sites suggested that the potential mRNA targets of the differentially expressed miRNAs may play important roles in muscle growth and development.

Figure 1. Reproducibility of the microarray experiments.

We examined the miRNA expression in three developmental stages of skeletal muscle (E33, E65 and Adu). Samples from each stage were isolated in triplicate and hybridized to the microarray. Scatter plots demonstrate the pair-wise comparison between each two sets of triplicates. The R represents the Spearman correlation coefficient.

Figure 2. Validation of the microarray results using Real-time PCR method.

Expression levels of five miRNAs (miR-150, miR-193b, miR-196a, miR-187b and miR-495) were detected by Real time PCR (right) and microarray (left). We have made a scaling to the raw data of Real time PCR to make it comparable to the microarray data. R represents the Pearson correlation coefficient.

Figure 3. Hierarchical cluster analysis.

We performed a data adjustment (median center and normalization) in the cluster analysis. The color codes of red, white, black and dark green represents expression levels of high, average, low and absent respectively. A detailed view of the miRNA expression levels in clustering patterns is shown in the plot areas from A to G.