Discovery of Porcine microRNAs in Multiple Tissues by a Solexa Deep Sequencing Approach

Abstract

The domestic pig (Sus scrofa) is an important economic animal for meat production and as a suitable model organism for comparative genomics and biomedical studies. In an effort to gain further identification of miRNAs in the pig, we have applied the Illumina Solexa sequencing technology to carry out an in-depth analysis of the miRNA transcriptome in a pool of equal amounts of RNA from 16 different porcine tissues. From this data set, we identified 437 conserved and 86 candidate novel miRNA/miRNA* in the pig, corresponding to 329 miRNA genes. Compared with all the reported porcine miRNAs, the result showed that 112 conserved and 61 candidate novel porcine miRNA were first reported in this study. Further analysis revealed extensive sequence variations (isomiRs) of porcine miRNAs, including terminal isomiRs at both the 5′ and 3′ ends and nucleotide variants. Read counts of individual porcine miRNA spanned from a few reads to approximately 405541 reads, confirming the different level of expression of porcine miRNAs. Subsequently, the tissue expression patterns of 8 miRNAs were characterized by Northern blotting. The results showed that miR-145, miR-423-5p, miR-320, miR-26a, and miR-191 are ubiquitously expressed in diverse tissues, while miR-92, miR-200a, and miR-375 were selectively enriched and expressed in special tissues. Meanwhile, the expression of 8 novel porcine-specific miRNAs was validated by stem-loop RT-PCR, and one of these was detected by Northern blotting. Using the porcine miRNA array designed according to our Solexa results, 123 miRNAs were detected expression in porcine liver tissues. A total of 58 miRNAs showed differential expression between the Tongcheng (a Chinese indigenous fatty breed) and Large White pig breeds (a lean type pig). Taken together, our results add new information to existing data on porcine miRNAs and should be useful for investigating the biological functions of miRNAs in pig and other species.

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

“…One study deciphered a high number of novel porcine miRNAs along the intestine and suggested a crucial role of miRNAs in intestinal functions (37). Another study examined the porcine miRNA transcriptome in different tissues and found that miR-423-5p was widely expressed (38). We confirmed the NGS results showing the upregulation of miR-423-5p in lymphocytes of ileal lymph nodes in piglets of the probiotic group at 1 week postweaning that were fed a diet with E. faecium.…”

Circulating microRNAs as diagnostic biomarkers for cardiovascular diseases

“…On the other hand, Goren et al (20) did find enrichment of miR-423-5p in the exosomal fraction of patients with HF, indicating that the mechanism of miR-423-5p release may be different in patients with HF compared with healthy controls. In the pig, miR-423-5p was shown to be ubiquitously expressed, with high levels in heart, liver, and brain (55). Together with the detected upregulation of miR-423-5p in human failing myocardium (41,42), this suggests that circulating miR-423-5p in HF is derived from the myocardium.…”

Solexa Sequencing of MicroRNAs on Chromium Metabolism in Broiler Chicks

“…Small RNA fractions were isolated from a 15% denaturing polyacrylamide gel to obtain 19- to 24-nt molecules, and 15 μg was ligated to 5′- and 3′-proprietary adaptors without de-phosphorylation and re-phosphorylation. The samples were reverse-transcribed into cDNA, which was amplified using RT-PCR following the Illumina protocol, and the DNA was sequenced on a Solexa machine (Illumina) [13,24]. Solexa sequencing was performed at the Beijing Genomics Institute (Beijing, China).…”