Expression stability of commonly used reference genes in canine articular connective tissues

<p>Abstract</p> <p>Background</p> <p>The quantification of gene expression in tissue samples requires the use of reference genes to normalise transcript numbers between different samples. Reference gene stability may vary between different tissues, and between the same tissue in different disease states. We evaluated the stability of 9 reference genes commonly used in human gene expression studies. Real-time reverse transcription PCR and a mathematical algorithm were used to establish which reference genes were most stably expressed in normal and diseased canine articular tissues and two canine cell lines stimulated with lipolysaccaride (LPS).</p> <p>Results</p> <p>The optimal reference genes for comparing gene expression data between normal and diseased infrapatella fat pad were <it>RPL13A </it>and <it>YWHAZ </it>(M = 0.56). The ideal reference genes for comparing normal and osteoarthritic (OA) cartilage were <it>RPL13A </it>and <it>SDHA </it>(M = 0.57). The best reference genes for comparing normal and ruptured canine cranial cruciate ligament were <it>B2M </it>and <it>TBP </it>(M = 0.59). The best reference genes for normalising gene expression data from normal and LPS stimulated cell lines were <it>SDHA </it>and <it>YWHAZ </it>(K6) or <it>SDHA </it>and <it>HMBS </it>(DH82), which had expression stability (M) values of 0.05 (K6) and 0.07 (DH82) respectively. The number of reference genes required to reduce pairwise variation (V) to <0.20 was 4 for cell lines, 5 for cartilage, 7 for cranial cruciate ligament and 8 for fat tissue. Reference gene stability was not related to the level of gene expression.</p> <p>Conclusion</p> <p>The reference genes demonstrating the most stable expression within each different canine articular tissue were identified, but no single reference gene was identified as having stable expression in all different tissue types. This study underlines the necessity to select reference genes on the basis of tissue and disease specific expression profile evaluation and highlights the requirement for the identification of new reference genes with greater expression stability for use in canine articular tissue gene expression studies.</p

Identification of new reference genes for the normalisation of canine osteoarthritic joint tissue transcripts from microarray data

<p>Abstract</p> <p>Background</p> <p>Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RT-qPCR) is the most accurate measure of gene expression in biological systems. The comparison of different samples requires the transformation of data through a process called normalisation. Reference or housekeeping genes are candidate genes which are selected on the basis of constitutive expression across samples, and allow the quantification of changes in gene expression. At present, no reference gene has been identified for any organism which is universally optimal for use across different tissue types or disease situations. We used microarray data to identify new reference genes generated from total RNA isolated from normal and osteoarthritic canine articular tissues (bone, ligament, cartilage, synovium and fat). RT-qPCR assays were designed and applied to each different articular tissue. Reference gene expression stability and ranking was compared using three different mathematical algorithms.</p> <p>Results</p> <p>Twelve new potential reference genes were identified from microarray data. One gene (mitochondrial ribosomal protein S7 [<it>MRPS7</it>]) was stably expressed in all five of the articular tissues evaluated. One gene HIRA interacting protein 5 isoform 2 [<it>HIRP5</it>]) was stably expressed in four of the tissues evaluated. A commonly used reference gene glyceraldehyde-3-phosphate dehydrogenase (<it>GAPDH</it>) was not stably expressed in any of the tissues evaluated. Most consistent agreement between rank ordering of reference genes was observed between <it>Bestkeeper©</it> and geNorm, although each method tended to agree on the identity of the most stably expressed genes and the least stably expressed genes for each tissue. New reference genes identified using microarray data normalised in a conventional manner were more stable than those identified by microarray data normalised by using a real-time RT-qPCR methodology.</p> <p>Conclusion</p> <p>Microarray data normalised by a conventional manner can be filtered using a simple stepwise procedure to identify new reference genes, some of which will demonstrate good measures of stability. Mitochondrial ribosomal protein S7 is a new reference gene worthy of investigation in other canine tissues and diseases. Different methods of reference gene stability assessment will generally agree on the most and least stably expressed genes, when co-regulation is not present.</p

Joint genomic prediction of canine hip dysplasia in UK and US Labrador Retrievers

Canine hip dysplasia, a debilitating orthopedic disorder that leads to osteoarthritis and cartilage degeneration, is common in several large-sized dog breeds and shows moderate heritability suggesting that selection can reduce prevalence. Estimating genomic breeding values require large reference populations, which are expensive to genotype for development of genomic prediction tools. Combining datasets from different countries could be an option to help build larger reference datasets without incurring extra genotyping costs. Our objective was to evaluate genomic prediction based on a combination of UK and US datasets of genotyped dogs with records of Norberg angle scores, related to canine hip dysplasia. Prediction accuracies using a single population were 0.179 and 0.290 for 1,179 and 242 UK and US Labrador Retrievers, respectively. Prediction accuracies changed to 0.189 and 0.260, with an increased bias of genomic breeding values when using a joint training set (biased upwards for the US population and downwards for the UK population). Our results show that in this study of canine hip dysplasia, little or no benefit was gained from using a joint training set as compared to using a single population as training set. We attribute this to differences in the genetic background of the two populations as well as the small sample size of the US dataset

Factors Associated with the Innate Orthopaedic Ability of Veterinary Students

Relatively little is known about the innate surgical ability of veterinary undergraduates. The objective of this study was to investigate if there were differences in the innate surgical ability of a cohort of 142 third-year veterinary undergraduate students to perform a series of simulated orthopaedic surgical tasks, and whether specific factors influenced their innate ability. Participants performed four simulated surgical tasks; ‘depth of plunge’ – an assessment of the ‘plunge’ depth through foam when drilling through the trans cortex of a PVC pipe; ‘3-dimensional drilling’ – an assessment of accuracy when drilling through a block of wood; ‘depth measurement’ – an assessment of the ability to correctly measure the depth of holes in PVC pipe; and ‘fracture reduction’ – where the speed and systematic reduction of a simulated fracture was assessed using a rubric score. Performance for each of these tasks was compared based on the responses to a survey. Results showed considerable variation in the innate ability of students. Previous experience performing manual tasks and using a drill was associated with an improvement in students’ ability to perform one of the four tasks (fracture reduction). Age, gender, handedness, videogame experience, building game experience, exposure to orthopaedic surgery, or desire to pursue surgery as a career were not associated with the performance of any of the tasks. A learning curve was observed for the depth of plunge task. An increased target angle led to decreased drilling accuracy for the 3D drilling task. The innate ability of veterinary students to undertake simulated surgical tasks was largely unaffected by the previous experiences we evaluated

Reductions in the Functional Diversity of Fish Communities in Certain Regions of the Northeast Shelf Regional Ecosystem Over 126 Years of Ocean Warming

&nbsp; Climate change has altered the functional diversity of fish communities around the globe through species distribution changes and increases in mortality. Reductions in functional diversity can deteriorate a community&rsquo;s resilience to future disturbance, as well as its ability to carry out ecosystem functions and services. As it has experienced a rate of warming faster than the rest of the globe&rsquo;s oceans, the fish communities of the Northeast Shelf Regional Ecosystem are particularly threatened by these potential changes. As has been seen around the globe in similarly large ecosystems, trends from this study suggest that some regions of the Northeast Shelf Regional Ecosystem will experience decreases in certain functional diversity metrics as warming continues, while others will experience increases in certain functional diversity metrics as warming continues. Additionally, trends suggest that ocean warming holds the potential to alter functional diversity in a relatively short period of time (here, in as little as 45 years). These findings can aid fisheries managers across the NSRE by allowing them to prioritize communities in the regions most at risk, and prepare for relatively fast fluctuations in resilience and ecosystem functioning. </p

The Genetic Basis of Canine Osteoarthritis

Osteoarthritis (OA) is a common debilitating disease ofmammalian joints. Canine OA was classically understood to arise secondary to articular diseases, such as elbow dysplasia, hip dysplasia and cranial cruciate ligament rupture which produced a mechanical dYllfunction of the affected joint. However it is now recognised that primary factors, such as genetics, govern the severity ofOA for a given articular disease. In this study, two different aspects ofthe genetic basis to OA were investigated; gene transcription in diseased tissues and gene polymorphism frequencies in populations of dogs with diseases predisposing to OA. Evaluation of the quality of extracted mRNA from canine joint tissues by rnicrofluidic electrophoresis traces revealed that there were no differences in the quality of samples extracted using either an isopropanol or ethanol precipitation method. However, a significant proportion ofRNA samples (32%) were identified as degraded, highlighting the importance of assessing RNA quality before usage. In OA canine hip cartilage, there was an increase in the gene expression of structural matrix molecules (collagens and small leucine rich proteoglycans) and proteinases (matrix metalloproteinase 13, cathepsin -B and -D), with concurrent decreased expression of selected inhibitors or protease activity (tissue inhibitors of metalloproteinase-2 and -4) when compared to normal articular cartilage using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analyses. The general pattern of changes in matrix-associated gene expression was similar to that reported in naturally occurring human OA cartilage. Canine-specific oligonucleotide microarray gene expression profiling of a small sample set of normal and OA articular cartilage samples identified differential expression of a number of genes not previously associated with the disease. However, the high degree of heterogeneity observed in the expression profile data generated hampered subsequent data interpretation, and highlighted the limitations of expression profiling small sample sets with limited phenotype stringency. Quantification of matrix-associated gene expression in OA elbow cartilage by RT-qPCR identified changes which were consistent with those identified in end stage hip OA cartilage, and which correlated with the radiographic severity of elbow OA for a number of genes (such as type I collagen alpha two chain, type III collagen alpha one chain and tissue inhibitor of matrix metalloproteinase 2). Matrix metalloproteinase expression in OA elbow trabecular bone was also identified to be increased when compared to normal trabecular bone, when quantified by RT-qPCR. A general pattern of increased protease and extracellular structural matrix gene expression was identified in ruptured canine cranial cruciate ligaments (CCL) when compared to intact CCLs, with both RTqPCR and oligonucleotide microarray. No significant differences were identified between the gene expression profiles of normal CCLs of a breed predisposed to CCL rupture (Labrador Retriever) when compared to a breed relatively resistant to CCL rupture (Greyhound), although a degree of risk-specific clustering was observed for expression profiles of genes which were differentially expressed in CCL rupture. The expression profiles ofruptured canine CCLs were similar to those previously reported for ruptured human CCLs. A transcriptomic basis to breed specific risk for the development ofcanine CCL rupture was not identified. Microarray data sets generated from normal and OA canine articular cartilage and normal and ruptured CCL were filtered to identify new reference genes for use in RT-qPCR experiments. One of the new reference genes (Mitochondrial ribosomal protein S7 [MRPS7]) demonstrated a high degree of stability across multiple articular tissues from normal and OA canine joints, as determined by multiple, different reference gene stability assessment algorithms, making it a potential universal reference gene for use in canine OA tissue studies. Silica membrane spin columns provided the most consistent recovery of high quantities of genomic DNA (gDNA) from EDTA preserved and clotted blood samples without the co-extraction of PCR inhibitors, when compared to phenol-chloroform or modified salt precipitation methods of DNA extraction. Spectrophotometer quantification of extracted gDNA did not provide an accurate assessment of the functional gDNA quantity with phenol-chloroform extracted samples, because ofprotein contamination. Single nucleotide polymorphisrns (SNPs) were identified in twenty candidate genes and their allele frequencies evaluated in populations of Labrador Retrievers and Golden Retrievers with cruciate ligament disease, populations of Labrador Retrievers with elbow dysplasia and hip dysplasia and compared to general populations of Labradors Retrievers and Golden Retrievers. Significant associations were identified for the minor allele and haplotype frequencies of SNPs in interieukin 12B (ILI2B) in Labrador Retrievers with elbow dysplasia, interieukin 4 (IL4) and interieukin 6 (IL6) in Labrador Retrievers with hip dysplasia, IL4 and ILl2B in Labrador Retrievers with cranial cruciate ligament rupture, and interieukin 10 (ILID) and Ankyrin repeat domain 10 (ANKRDIO) in Golden Retrievers with cranial cruciate ligament rupture. A common genomic risk for the articular disease, or OA, was not identified between the two different breeds of dog evaluated, but common genomic risks were identified for different articular diseases within a single breed. A genetic basis to canine articular disease, or OA, was confirmed

Dogslife: A web-based longitudinal study of Labrador Retriever health in the UK

<p>Abstract</p> <p>Background</p> <p>Dogslife is the first large-scale internet-based longitudinal study of canine health. The study has been designed to examine how environmental and genetic factors influence the health and development of a birth cohort of UK-based pedigree Labrador Retrievers.</p> <p>Results</p> <p>In the first 12 months of the study 1,407 Kennel Club (KC) registered eligible dogs were recruited, at a mean age of 119 days of age (SD 69 days, range 3 days – 504 days). Recruitment rates varied depending upon the study team’s ability to contact owners. Where owners authorised the provision of contact details 8.4% of dogs were recruited compared to 1.3% where no direct contact was possible. The proportion of dogs recruited was higher for owners who transferred the registration of their puppy from the breeder to themselves with the KC, and for owners who were sent an e-mail or postcard requesting participation in the project. Compliance with monthly updates was highly variable. For the 280 dogs that were aged 400 days or more on the 30^th June 2011, we estimated between 39% and 45% of owners were still actively involved in the project. Initial evaluation suggests that the cohort is representative of the general population of the KC registered Labrador Retrievers eligible to enrol with the project. Clinical signs of illnesses were reported in 44.3% of Labrador Retrievers registered with Dogslife (median age of first illness 138 days), although only 44.1% of these resulted in a veterinary presentation (median age 316 days).</p> <p>Conclusions</p> <p>The web-based platform has enabled the recruitment of a representative population of KC registered Labrador Retrievers, providing the first large-scale longitudinal population-based study of dog health. The use of multiple different methods (e-mail, post and telephone) of contact with dog owners was essential to maximise recruitment and retention of the cohort.</p