Microarray-based approach identifies differentially expressed microRNAs in porcine sexually immature and mature testes

Abstract

Background: MicroRNAs (miRNAs) are short non-coding RNA molecules which are proved to be involved in mammalian spermatogenesis. Their expression and function in the porcine germ cells are not fully understood.

Methodology: We employed a miRNA microarray containing 1260 unique miRNA probes to evaluate the miRNA expression patterns between sexually immature (60-day) and mature (180-day) pig testes. One hundred and twenty nine miRNAs representing 164 reporter miRNAs were expressed differently (p<0.1). Fifty one miRNAs were significantly up-regulated and 78 miRNAs were down-regulated in mature testes. Nine of these differentially expressed miRNAs were validated using quantitative RT-PCR assay. Totally 15,919 putative miRNA-target sites were detected by using RNA22 method to align 445 NCBI pig cDNA sequences with these 129 differentially expressed miRNAs, and seven putative target genes involved in spermatogenesis including DAZL, RNF4 gene were simply confirmed by quantitative RT-PCR.

Conclusions: Overall, the results of this study indicated specific miRNAs expression in porcine testes and suggested that miRNAs had a role in regulating spermatogenesis.

Figure 1. Validation of the microarray results using qRT-PCR method.

The X-axis represents the miRNAs and the Y-axis shows the relative expression levels of miRNAs (-ΔC_t values for qRT-PCR; Log(sample signal, 2) for microarray). The number of biological replicates is three for both assays. R represents the Pearson correlation coefficient. The significance of differences for the expression between 60-d (immature, 60-day) and 180-d (mature, 180-day) testes was calculated using two-tailed T-test. *, p≤0.05; **, p≤0.01 (left for qRT-PCR, and right for microarray).

Figure 2. miRNA putative target genes expression in porcine testis was identified by qRT-PCR.

The X-axis represents the specific gene and the Y-axis shows the ΔΔC_t values of genes. ΔΔC_t value has a negative relationship with the gene expression level, so the smaller ΔΔC_t value has a higher expression level. The number of biological replicates is three. The significance of differences for the expression between 60-d (immature, 60-day) and 180-d (mature, 180-day) testes was calculated using two-tailed T-test. *, p≤0.05; **, p≤0.01.