ABSTRACT
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.
Subject(s)
Dogs/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Animals , Humans , MicroRNAs/analysis , Nucleic Acid Amplification Techniques , Sequence Analysis, RNA , Tissue DistributionABSTRACT
OBJECTIVE: To identify microsatellite markers linked to progressive retinal atrophy (PRA) in American Eskimo Dogs. SAMPLE POPULATION: Blood samples or buccal epithelial cells from 66 American Eskimo Dogs, including 53 PRA-unaffected and 13 PRA-affected dogs. PROCEDURE: The genotypes of unaffected and affected dogs were determined by use of microsatellite markers spanning canine chromosome 9 (CFA09). Homozygosity mapping was used to detect linkage between markers and the gene locus for PRA. RESULTS: Significant allelic association between marker alleles and the gene locus for PRA was detected for GALK1 and TK1, indicating linkage between these markers and the causative gene locus for PRA. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that PRA in American Eskimo Dogs is located on CFA09 and allow for the development of a microsatellite-based test to identify carrier (unaffected) and affected dogs before clinical signs appear.
