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Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint
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Suitable reference gene selection for gene expression studies in knee osteoarthritis synovium using quantitative PCR
Hiroshi Watanabe1,2, Hirotaka Ishii1, Kenji Takahashi2
1a Department of Anatomy and Neurobiology , Graduate School of Medicine, Nippon Medical School , Bunkyo-ku , Tokyo , Japan.
Connective Tissue Research
|November 3, 2017
Summary
For accurate gene expression analysis in osteoarthritis (OA) synovium, HPRT1 is the best reference gene. Using the wrong reference gene, like B2M, can alter study results and significance.
Area of Science:
- Molecular Biology
- Genetics
- Rheumatology
Background:
- Osteoarthritis (OA) is a primary cause of disability in older adults.
- Gene expression profiling via RT-qPCR is crucial for understanding OA's molecular features.
- Accurate normalization using stable reference genes is essential for reliable RT-qPCR results.
Purpose of the Study:
- To identify the most stable reference gene for normalizing RT-qPCR data in knee osteoarthritis (KOA) synovium.
- To evaluate the expression of steroid hormone receptors and proinflammatory cytokines in KOA synovium.
- To assess the impact of reference gene selection on the interpretation of gene expression data and correlations with KOA risk factors.
Main Methods:
- Total RNA was isolated from synovium samples of 38 patients undergoing total knee arthroplasty for KOA.
- Expression stability of seven candidate housekeeping genes (ACTB, B2M, GAPDH, HPRT1, RPL13A, SDHA, YWHAZ) was validated using geNorm, NormFinder, and BestKeeper algorithms.
- Expression profiles of steroid hormone receptor genes (AR, ESR1, ESR2, GR, MR, PR) and proinflammatory cytokine genes (IL1B, IL6) were analyzed.
Main Results:
- HPRT1 was identified as the most stable housekeeping gene, while B2M was the least stable in KOA synovium.
- Sexually dimorphic expression patterns were observed for AR, IL1B, and IL6.
- The choice of reference gene significantly impacted the observed correlations between gene expression, risk factors (e.g., BMI), and statistical significance.
Conclusions:
- HPRT1 is the recommended reference gene for accurate normalization in RT-qPCR studies of OA synovium.
- Inappropriate reference gene selection can lead to erroneous conclusions regarding gene expression and disease associations.
- The study highlights the importance of reference gene validation for robust molecular characterization of osteoarthritis.
