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Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering
Tao He1,2, Yijiang Huang1, Juy Chi Chak1
1Laboratory of Biomechanics and Experimental Orthopaedics, Department of Orthopaedic Surgery, Physical Medicine and Rehabilitation, University Hospital of Munich (LMU), Munich, Germany.
Scientific Reports
|October 7, 2018
Summary
Reference gene stability is crucial for accurate gene expression analysis in tissue engineering. Always validate reference genes for quantitative reverse transcription polymerase chain reaction (qRT-PCR) in each new experiment to ensure reliable results.
Area of Science:
- Biomaterials Science
- Molecular Biology
- Regenerative Medicine
Background:
- Autogenous tissue grafting is standard for bone and cartilage defects.
- Translating tissue-engineered osteogenesis and chondrogenesis to clinical practice faces challenges.
- Imprecise gene expression profiling due to non-standardized quantitative gene assays is an underestimated barrier.
Purpose of the Study:
- To assess reference gene stability for quantitative reverse transcription polymerase chain reaction (qRT-PCR) in tissue engineering.
- To determine optimal cDNA amounts for accurate gene expression analysis.
- To identify potential variations in reference gene stability across different cell and tissue types.
Main Methods:
- Utilized GeNorm algorithm for reference gene stability analysis.
- Assessed gene expression in human bone marrow-derived mesenchymal stem cells (hMSCs) and adipose-derived stem cells (ASCs).
- Included differentiated chondrocytes, normal human chondrocytes, and rat muscle tissue.
- Evaluated stability concerning varying cDNA amounts and experimental conditions.
Main Results:
- Reference gene stability varied significantly across different cell lines, tissue types, and experimental conditions.
- No universal set of reference genes is suitable for all experimental contexts in bone and cartilage tissue engineering.
- Optimal cDNA quantities for normalization were not consistent across all tested samples.
Conclusions:
- Reference gene stability and quantity are not conserved across diverse cell lines or tissue types.
- Tissue engineers using qRT-PCR must re-evaluate reference gene stability and normalization for each new experimental setup.
- Standardizing reference gene selection is critical for reproducible gene expression profiling in regenerative medicine research.
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