Identification of novel and differentially expressed MicroRNAs of dairy goat mammary gland tissues using solexa sequencing and bioinformatics

Abstract

MicroRNAs are small, noncoding RNA molecules that regulate gene expression at the post-transcriptional level and play an important role in various biological processes. Although most microRNAs expression profiles studies have been performed in humans or rodents, relatively limited knowledge also exists in other mammalian species. The identification of the full repertoire of microRNAs expressed in the lactating mammary gland of Capra hircus would significantly increase our understanding of the physiology of lactating mammary glands. In this study, two libraries were constructed using the lactating mammary gland tissues of Laoshan dairy goats (Capra hircus) during peak and late lactation. Solexa high-throughput sequencing technique and bioinformatics were used to determine the abundance and differential expression of the microRNAs between peak and late lactation. As a result, 19,044,002 and 7,385,833 clean reads were obtained, respectively, and 1,113 conserved known microRNAs and 31 potential novel microRNA candidates were identified. A total of 697 conserved microRNAs were significantly differentially expressed with a P-value<0.01, 272 microRNAs were up-regulated and 425 microRNAs were down-regulated during peak lactation. The results were validated using real-time quantitative RT-PCR. 762,557 annotated mRNA transcripts were predicted as putative target gene candidates. The GO annotation and KEGG pathway analysis suggested that differentially expressed microRNAs were involved in mammary gland physiology, including signal transduction, and cell-cell and cell-extracellular communications. This study provided the first global of the microRNA in Capra hircus and expanded the repertoire of microRNAs. Our results have great significance and value for the elucidation of complex regulatory networks between microRNAs and mRNAs and for the study of mammary gland physiology and lactation.

Figure 1. Length distribution and abundance of sequences in peak and late lactation.

Sequence length distribution of clean reads based on the abundance and distinct sequences; the most abundant size class was 22 nt, followed by 20 nt, 23 nt and 21 nt.

Figure 2. Distribution of small RNAs among different categories in peak and late lactation.

The clean reads were annotated and classified as miRNA, rRNA, tRNA, snRNA and snoRNA in GenBank and Rfam databases, and partial reads were not annotated.

Figure 3. Comparison of expression levels of miRNAs in peak and late lactation.

The X- and Y-axes show the expression levels of miRNAs in the two samples. The red points represent miRNAs with ratios >2; the blue points represent miRNAs with 1/2

Figure 4. qRT-PCR validation of the identified miRNAs using Solexa sequencing technology.

A: Real-time RT-PCR results for 9 known miRNAs in peak and late lactation. The relative quantification of expression was calculated using the 2^−△△CT method after the threshold cycle (Ct) and was normalized with the Ct of U6. The relative expression levels were presented as the 2^−△△Ct means ± SE. The error bars indicate the standard error of the 2^−△△Ct mean values. * represents p<0.05, ** represents p<0.01. B: Potential novel miRNAs expression in mammary gland. Total RNA pooled from five individuals of two lactation periods were used for qRT-PCR, the relative expression abundance were expressed as Ct value, each sample was replicated for three times.

Figure 5. Part gene ontology classification annotated by gene2go for predicted target genes.

The figure shows partial GO enrichment for the predicted target genes in molecular function, cellular component and biological processes.