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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.
Abstract: 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. Methods: 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. Methods: 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.
Publication Date: 2000-08-22 PubMed ID: 10951979DOI: 10.2460/ajvr.2000.61.900Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Comparative Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research article presents a comparative study of the use of Northern blot hybridization and a reverse transcriptase-polymerase chain reaction (RT-PCR) technique for measuring mRNA expression of certain proteins and components involved in osteoarthritis in horses.
About The Research
- In this study, researchers are attempting to accurately measure the mRNA expression of aggrecan, type-II collagen, matrix metalloproteinase (MMP) 1, and MMP3. These components play crucial roles in joint health and are often associated with conditions such as osteoarthritis (OA).
- The subjects were articular cartilage and synovial membrane samples derived from eight pairs of equine joints, with one joint with osteoarthritis and one healthy pair from each of six horses.
- Additionally, synovial membrane samples were also gathered from six pairs of joints from five horses. Articular cartilage is a connective tissue that coats the surface of joints, while synovial membrane encloses the joint cavity and secretes synovial fluid that reduces friction in joints during movement.
- The overall objective was to compare the efficiency of Northern blot hybridization with that of a RT-PCR assay in quantifying mRNA expression of the aforementioned components.
Methods and Findings
- The researchers extracted RNA from the joint samples using a modified Trizol procedure, and then applied Northern blot hybridization and RT-PCR.
- They found that articular cartilage samples from joints with mild or moderate osteoarthritis yielded less total RNA than samples from joints with severe OA.
- Northern blot hybridization revealed that type-II collagen mRNA expression in OA cartilage samples was significantly higher than in healthy samples. However, when using the RT-PCR assay, they were able to identify low levels of MMP3 mRNA expression in four sets of articular cartilage and synovial membrane samples.
- On the contrary, Northern blot hybridization only detected MMP3 mRNA expression in one set of the same samples. This significant variance in results led the researchers to conclude that the RT-PCR assay is more sensitive than Northern blot hybridization, and additionally requires smaller samples.
Implications of the Findings
- The higher sensitivity of the RT-PCR assay in detecting mRNA expressions gives it a clear advantage over Northern blot hybridization, as it may allow for earlier detection of osteoarthritis through monitoring shifts in the expression levels of aggrecan, type-II collagen, MMP1, and MMP3.
- Moreover, requiring smaller samples makes RT-PCR assay a less invasive method, which is important in the field of veterinary medicine, for the welfare of the animals under study.
Cite This Article
APA
Fehr JE, Trotter GW, Oxford JT, Hart DA.
(2000).
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.
Am J Vet Res, 61(8), 900-905.
https://doi.org/10.2460/ajvr.2000.61.900 Publication
Researcher Affiliations
- Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins 80523, USA.
MeSH Terms
- Aggrecans
- Animals
- Blotting, Northern / veterinary
- Cartilage, Articular / chemistry
- Cartilage, Articular / pathology
- Collagen / analysis
- Collagen / biosynthesis
- Collagen / physiology
- DNA Primers / chemistry
- Extracellular Matrix Proteins / analysis
- Extracellular Matrix Proteins / biosynthesis
- Extracellular Matrix Proteins / physiology
- Gene Expression Regulation
- Horse Diseases / pathology
- Horses
- Image Processing, Computer-Assisted
- Joints / pathology
- Lectins, C-Type
- Matrix Metalloproteinase 1 / analysis
- Matrix Metalloproteinase 1 / biosynthesis
- Matrix Metalloproteinase 1 / physiology
- Matrix Metalloproteinase 3 / analysis
- Matrix Metalloproteinase 3 / biosynthesis
- Matrix Metalloproteinase 3 / physiology
- Matrix Metalloproteinases / analysis
- Matrix Metalloproteinases / biosynthesis
- Matrix Metalloproteinases / physiology
- Osteoarthritis / pathology
- Osteoarthritis / veterinary
- Proteoglycans / analysis
- Proteoglycans / biosynthesis
- Proteoglycans / physiology
- RNA, Messenger / chemistry
- RNA, Messenger / isolation & purification
- Reverse Transcriptase Polymerase Chain Reaction / veterinary
- Synovial Membrane / chemistry
- Synovial Membrane / pathology
Citations
This article has been cited 15 times.- Liharska L, Charney A. Transcriptomics : Approaches to Quantifying Gene Expression and Their Application to Studying the Human Brain. Curr Top Behav Neurosci 2024;68:129-176.
- Pagani S, Maglio M, Sicuro L, Fini M, Giavaresi G, Brogini S. RNA Extraction from Cartilage: Issues, Methods, Tips. Int J Mol Sci 2023 Jan 20;24(3).
- Sikora M, Krajewska K, Marcinkowska K, Raciborska A, Wiglusz RJ, Śmieszek A. Comparison of Selected Non-Coding RNAs and Gene Expression Profiles between Common Osteosarcoma Cell Lines. Cancers (Basel) 2022 Sep 19;14(18).
- Tonyan ZN, Nasykhova YA, Danilova MM, Barbitoff YA, Changalidi AI, Mikhailova AA, Glotov AS. Overview of Transcriptomic Research on Type 2 Diabetes: Challenges and Perspectives. Genes (Basel) 2022 Jun 30;13(7).
- Yang X, Lou H, Chen YT, Huang SF, Loh YP. A novel 40kDa N-terminal truncated carboxypeptidase E splice variant: cloning, cDNA sequence analysis and role in regulation of metastatic genes in human cancers. Genes Cancer 2019;10(5-6):160-170.
- Cai J, Li P, Luo X, Chang T, Li J, Zhao Y, Xu Y. Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barley. PLoS One 2018;13(1):e0190559.
- Samadi R, Shafiei B, Azizi F, Ghasemi A. Radioactive Iodine Therapy and Glucose Tolerance. Cell J 2017 Jul-Sep;19(2):184-193.
- Speziali A, Delcogliano M, Tei M, Placella G, Chillemi M, Tiribuzi R, Cerulli G. Chondropenia: current concept review. Musculoskelet Surg 2015 Dec;99(3):189-200.
- Xi D, He Y, Sun Y, Gou X, Yang S, Mao H, Deng W. Molecular cloning, sequence identification and tissue expression profile of three novel genes Sfxn1, Snai2 and Cno from Black-boned sheep (Ovis aries). Mol Biol Rep 2011 Mar;38(3):1883-7.
- Yang L, He Y, Kong Q, Zhang W, Xi D, Mao H, Deng W. Isolation, nucleotide identification and tissue expression of three novel ovine genes-SLC25A4, SLC25A5 and SLC25A6. Mol Biol Rep 2010 Jul;37(6):2743-8.
- Liu GY, Xiong YZ. Molecular cloning and expression analyses of a novel swine gene--ARF4. Mol Biol Rep 2009 Mar;36(3):455-60.
- Liu GY, Xiong YZ. Molecular characterization and expression profile of a novel porcine gene differentially expressed in the muscle tissues from Meishan, Large White and their hybrids. Mol Biol Rep 2009 Jan;36(1):57-62.
- Liu GY, Gao SZ, Ge CR, Zhang X. Molecular characterization of the encoding regions and tissue expression analyses for three novel porcine genes--HNRPA1, YIPF5 and UB2D2. Mol Biol Rep 2008 Dec;35(4):519-26.
- Liu GY, Ge CR, Zhang X, Gao SZ. Isolation, sequence identification and tissue expression distribution of three novel porcine genes--RAB14, S35A3 and ITM2A. Mol Biol Rep 2008 Jun;35(2):201-6.
- Liu GY, Xiong YZ. Isolation, sequence analysis and expression profile of a novel porcine gene, NIP7, differentially expressed in the Longissimus dorsi muscle tissues from Meishan, Meishan x Large White cross and Large White pigs. Mol Biol Rep 2007 Dec;34(4):213-9.
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