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Comparison of Northern blot hybridization and a reverse transcriptase-polymerase chain reaction technique for measurement of mRNA expression of metalloproteinases and matrix components in articular cartilage and synovial membrane from horses with osteoarthritis

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  • 1 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.
  • | 2 present address is 3549 254th Ave SE, Issaquah, WA 98029.
  • | 3 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.
  • | 4 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.
  • | 5 McCraig Center for Joint Injury and Arthritis Research, Faculty of Medicine, University of Calgary, Calgary, Alberta, T2N 4N1.

Abstract

Objective—To determine relative amounts of mRNA expression of aggrecan, type-II collagen, matrix metalloproteinase (MMP) 1, and MMP3 in articular cartilage and synovial membrane samples from healthy equine joints and joints with osteoarthritis (OA) and to compare results of Northern blot hybridization with results of a reverse transcriptase-polymerase chain reaction (RT-PCR) assay.

Sample Population—Articular cartilage samples from 8 pairs of joints (1 with OA and 1 healthy) from 6 horses and synovial membrane samples from 6 pairs of joints from 5 horses.

Procedure—RNA was extracted from samples by use of a modified Trizol procedure. Northern blot hybridization and the RT-PCR assay were performed; results were quantitated by use of glyceraldehyde 3- phosphate dehydrogenase as an internal standard.

Results—Articular cartilage samples from joints with mild or moderate OA yielded less total RNA than samples from joints with severe OA. Northern blot hybridization indicated that type-II collagen mRNA expression in articular cartilage samples from joints with OA was significantly greater than expression in samples from healthy joints. The RT-PCR assay identified low levels of MMP3 mRNA expression in 4 of 8 sets of articular cartilage samples and 4 of 6 sets of synovial membrane samples, whereas Northern blot hybridization identified MMP3 mRNA expression in only 1 of 6 sets of articular cartilage samples and 1 of 6 sets of synovial membrane samples.

Conclusions—A RT-PCR assay is more sensitive than Northern blot hybridization for detection of MMP3 mRNA expression in articular cartilage and synovial membrane and requires smaller samples. (Am J Vet Res 2000;61:900–905)

Abstract

Objective—To determine relative amounts of mRNA expression of aggrecan, type-II collagen, matrix metalloproteinase (MMP) 1, and MMP3 in articular cartilage and synovial membrane samples from healthy equine joints and joints with osteoarthritis (OA) and to compare results of Northern blot hybridization with results of a reverse transcriptase-polymerase chain reaction (RT-PCR) assay.

Sample Population—Articular cartilage samples from 8 pairs of joints (1 with OA and 1 healthy) from 6 horses and synovial membrane samples from 6 pairs of joints from 5 horses.

Procedure—RNA was extracted from samples by use of a modified Trizol procedure. Northern blot hybridization and the RT-PCR assay were performed; results were quantitated by use of glyceraldehyde 3- phosphate dehydrogenase as an internal standard.

Results—Articular cartilage samples from joints with mild or moderate OA yielded less total RNA than samples from joints with severe OA. Northern blot hybridization indicated that type-II collagen mRNA expression in articular cartilage samples from joints with OA was significantly greater than expression in samples from healthy joints. The RT-PCR assay identified low levels of MMP3 mRNA expression in 4 of 8 sets of articular cartilage samples and 4 of 6 sets of synovial membrane samples, whereas Northern blot hybridization identified MMP3 mRNA expression in only 1 of 6 sets of articular cartilage samples and 1 of 6 sets of synovial membrane samples.

Conclusions—A RT-PCR assay is more sensitive than Northern blot hybridization for detection of MMP3 mRNA expression in articular cartilage and synovial membrane and requires smaller samples. (Am J Vet Res 2000;61:900–905)