Identification, amplification and characterization of miR-17-92 from canine tissue. Gene

Department of Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA.
Gene (Impact Factor: 2.14). 01/2008; 404(1-2):25-30. DOI: 10.1016/j.gene.2007.08.015
Source: PubMed


Recently, a novel group of genes encoding small RNA molecules, termed microRNAs (miRNAs), has been discovered to play a vital role in eukaryotic gene expression. Known to act in a post-transcriptional fashion, miRNAs can inhibit translation by binding to messenger RNA (mRNA) or by targeting mRNA for degradation. A search of genetic databases revealed significant conservation of miRNA genes between the domestic dog and the human. This finding suggests that expression patterns may also be conserved. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan Real-time PCR techniques were used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver) using the delta-delta Ct (critical threshold) method.

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    • "Human and canine data were normalized compared with the endogenous reference RNU44, following Applied Biosystems indications (TaqMan miRNA assay n°4373384 – PE Applied Biosystems). We also tested RNU6B, an endogenous reference previously used in dogs (Boggs et al., 2007, 2008) but RNU44 was chosen as it proved to be more constant and reliable. Each was analysed in duplicate. "
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    ABSTRACT: Osteosarcoma (OS) is the most common primary malignant bone tumour in dogs and humans. MicroRNAs are short non-coding RNA molecules involved in post-transcriptional gene expression. Here, we compared the effects of miR-196a deregulation in human and canine OS cells after having observed a more uniform distribution and stronger down-expression in the human specimens. Cell response to miR-196a transfection was different in human and canine OS. A decreased proliferation rate was seen in human MG63 and 143B OS cells, while no appreciable changes occurred in canine DAN cells. Transient decrease of motility was highly remarkable and longer in MG63, concomitant with decreased levels of annexin1, a target of miR-196a promoting cell migration and invasion.
    Full-text · Article · Dec 2014 · Research in Veterinary Science
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    • "Consistent with a published study on horse miRNAs [18], a miR-17-92 cluster was also detected on chromosome 17 in our study. This cluster is reportedly harboring miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a, and is a well-preserved cluster in human and all vertebrates [11]–[18]. In mammals, the miR-17-92 cluster is divided into two cluster paralogs: miR-106b-25 cluster and the miR-106a-363 cluster [47]. "
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    ABSTRACT: The role of microRNAs (miRNAs) as a post-transcriptional gene regulator has been elucidated in a broad range of organisms including domestic animals. Characterization of miRNAs in normal tissues is an important step to investigate the functions of miRNAs in various physiological and pathological conditions. Using Illumina Next Generation Sequencing (NGS) technology, we identified a total of 292 known and 329 novel miRNAs in normal horse tissues including skeletal muscle, colon and liver. Distinct sets of miRNAs were differentially expressed in a tissue-specific manner. The miRNA genes were distributed across all the chromosomes except chromosomes 29 and 31 in the horse reference genome. In some chromosomes, multiple miRNAs were clustered and considered to be polycistronic transcript. A base composition analysis showed that equine miRNAs had a higher frequency of A+U than G+C. Furthermore, U tended to be more frequent at the 5' end of miRNA sequences. This is the first experimental study that identifies and characterizes the global miRNA expression profile in normal horse tissues. The present study enriches the horse miRNA database and provides useful information for further research dissecting biological functions of miRNAs in horse.
    Full-text · Article · Apr 2014 · PLoS ONE
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    • "Expression levels of seven miRNAs were analyzed in several canine tissues. This was the first study that successfully used quantitative Real-Time RT-PCR (qPCR) assays originally designed for human miRNA detection and showed full conservation of mature sequences of canine and human miRNAs [61]. In 2008, Zhou et al. identified 357 miRNA candidates from the dog genome, 300 of which were orthologous to already characterized human miRNAs [60]. "
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    ABSTRACT: Background Dilated cardiomyopathy (DCM) is the most common heart disease in Doberman Pinschers. MicroRNAs (miRNAs) are short non-coding RNAs playing important roles in gene regulation. Different miRNA expression patterns have been described for DCM in humans and might represent potential diagnostic markers. There are no studies investigating miRNA expression profiles in canine DCM. The aims of this study were to screen the miRNA expression profile of canine serum using miRNA microarray and to compare expression patterns of a group of Doberman Pinschers with DCM and healthy controls. Results Eight Doberman Pinschers were examined by echocardiography and 24-hour-ECG and classified as healthy (n = 4) or suffering from DCM (n = 4). Total RNA was extracted from serum and hybridized on a custom-designed 8x60k miRNA microarray (Agilent) containing probes for 1368 individual miRNAs. Although total RNA concentrations were very low in serum samples, 404 different miRNAs were detectable with sufficient signal intensity on miRNA microarray. 22 miRNAs were differentially expressed in the two groups (p < 0.05 and fold change (FC) > 1.5), but did not reach statistical significance after multiple testing correction (false discovery rate adjusted p > 0.05). Five miRNAs were selected for further analysis using quantitative Real-Time RT-PCR (qPCR) assays. No significant differences were found using specific miRNA qPCR assays (p > 0.05). Conclusions Numerous miRNAs can be detected in canine serum. Between healthy and DCM dogs, miRNA expression changes could be detected, but the results did not reach statistical significance most probably due to the small group size. miRNAs are potential new circulating biomarkers in veterinary medicine and should be investigated in larger patient groups and additional canine diseases.
    Full-text · Article · Jan 2013 · BMC Veterinary Research
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