Dataset Information

Analysis of normal and osteoarthritic canine cartilage mRNA expression by quantitative polymerase chain reaction.

ABSTRACT: The molecular basis to mammalian osteoarthritis (OA) is unknown. We hypothesised that the expression of selected proteases, matrix molecules, and collagens believed to have a role in the pathogenesis of OA would be changed in naturally occurring canine OA cartilage when compared to normal articular cartilage. Quantitative (real-time) reverse transcriptase-polymerase chain reaction assays were designed measuring the expression of selected matrix molecules (collagens and small leucine-rich proteoglycans), key mediators of the proteolytic degradation of articular cartilage (metalloproteinases, cathepsins), and their inhibitors (tissue inhibitors of matrix metalloproteinases). All data were normalised using a geometric mean of three housekeeping genes, and the results subjected to power calculations and corrections for multiple hypothesis testing. We detected increases in the expression of BGN, COL1A2, COL2A1, COL3A1, COL5A1, CSPG2, CTSB, CTSD, LUM, MMP13, TIMP1, and TNC in naturally occurring canine OA. The expression of TIMP2 and TIMP4 was significantly reduced in canine OA cartilage. The patterns of gene expression change observed in naturally occurring canine OA were similar to those reported in naturally occurring human OA and experimental canine OA. We conclude that the expression profiles of matrix-associated molecules in end-stage mammalian OA may be comparable but that the precise aetiologies of OA affecting specific joints in different species are presently unknown.

SUBMITTER: Clements DN

PROVIDER: S-EPMC1794499 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Analysis of normal and osteoarthritic canine cartilage mRNA expression by quantitative polymerase chain reaction.

Clements Dylan N DN Carter Stuart D SD Innes John F JF Ollier William E R WE Day Philip J R PJ

Arthritis research & therapy 20060101 6

The molecular basis to mammalian osteoarthritis (OA) is unknown. We hypothesised that the expression of selected proteases, matrix molecules, and collagens believed to have a role in the pathogenesis of OA would be changed in naturally occurring canine OA cartilage when compared to normal articular cartilage. Quantitative (real-time) reverse transcriptase-polymerase chain reaction assays were designed measuring the expression of selected matrix molecules (collagens and small leucine-rich proteog ...[more]

PMID: 17032449

Similar Datasets

Project description:A quantitative polymerase chain reaction (PCR) method for determining concentrations of mRNA for the cyclic AMP (cAMP)-binding protein RI alpha, a regulatory subunit of cAMP-dependent protein kinase, was developed using site-directed mutagenic primers and mix-melt PCR. The PCR product for RI alpha mRNA was modified to include an EcoRV restriction site for use as an internal standard. This mutant utilised the same primers as the target mRNA and differed in sequence by only four bases. As only one of these base changes results in a purine/pyrimidine switch the effective change in labelling with [32P]dCTP was less than 0.5%. Reverse transcription of mRNA was performed and quantitative PCR was carried out using fixed levels of mutant RI alpha vs varying amounts of both normal RI alpha sequence of known concentration and unknown samples. Validation of the technique using rigourous quality control established that reverse transcription, determined by incorporation of labelled nucleotides, gave intra- and interassay variations of 16.2 and 9.3% respectively. Using crossover evaluation of cDNA concentrations with cloned RI alpha sequences as controls intra- and interassay variations of 14.3% and 4-8% respectively were obtained. Using compounded errors, the limits of precision for this technique demonstrate that values that are altered by 50% or more represent a true alteration in mRNA levels between samples tested. This value compares favourably to similar values for radioimmunoassays of between 10% and 30% precision. Analysis of a series of patient samples during routine follow-up of treatment demonstrated clear changes in mRNA levels. Using site-directed mutagenesis to establish a quantitative PCR-based assay for expression of mRNA this study demonstrates the potential usefulness and some limitations of quantitative PCR for applications within a clinical biochemistry laboratory. However, based on compounded error, values that vary by less than 50% within assays, and by less than 70% in separate assays could not be clearly separated. Assessment of paired patient samples has demonstrated clear changes in mRNA for the target protein RI alpha. With the use of normal quality control procedures this study has established that the degree of reproducibility of this quantitative PCR technique would allow assessment of mRNA levels for markers of interest in clinical samples in a routine laboratory context.

Dataset Information

Analysis of normal and osteoarthritic canine cartilage mRNA expression by quantitative polymerase chain reaction.

Publications

Analysis of normal and osteoarthritic canine cartilage mRNA expression by quantitative polymerase chain reaction.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure