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Validation of reference genes in human chordoma
R G C Santegoeds1, Y Yakkioui1, A Jahanshahi1
1Department of Neurosurgery, School for Mental Health and Neuroscience, Maastricht University Medical Centre, Maastricht, The Netherlands.
Background:
Chordoma are rare slow-growing tumors of the axial skeleton, which are thought to arise from remnants of the notochord. Little is known about the underlying mechanisms that drive this tumor. However, the assessment of gene expression levels by quantitative real-time polymerase chain reaction (qRT-PCR) is hampered due to a lack of validated reference genes. Using an unstable reference gene in qRT-PCR may lead to irreproducible results.
Methods:
The expression of 12 candidate reference genes (ACTB, B2M, T, EF1a, GAPDH, HPRT, KRT8, KRT19, PGK1, RS27a, TBP, and YWHAZ) was analyzed by qRT-PCR in flash frozen chordoma samples from 18 patients. GeNorm and NormFinder algorithms were used to rank the stability of the genes.
Results:
From most to least stably expressed, the top six genes found by geNorm were PGK1, YWHAZ, ACTB, HPRT, EF1A, and TBP. When analyzed by NormFinder, the top six genes were ACTB, YWHAZ, PGK1, B2M, TBP, and HPRT. GAPDH alone, which is often used as a reference gene in chordoma gene expression studies, is not stable enough for reliable results.
Conclusion:
In gene expression studies of human chordomas, PGK1, ACTB, and YWHAZ are more stably expressed, and therefore, are preferred reference genes over the most often used reference gene so far, GAPDH.
Insights
Identifying stable reference genes is crucial for accurate chordoma gene expression studies. PGK1, ACTB, and YWHAZ are recommended over GAPDH for reliable qRT-PCR results in chordoma research.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Chordomas are rare, slow-growing tumors originating from the axial skeleton, likely from notochord remnants.
- The molecular mechanisms driving chordoma development remain largely unknown.
- Accurate gene expression analysis using quantitative real-time polymerase chain reaction (qRT-PCR) is hindered by the absence of validated reference genes, potentially leading to unreliable findings.
Purpose of the Study:
- To identify and validate the most stable reference genes for gene expression studies in human chordoma.
- To compare the stability of commonly used and novel candidate reference genes.
- To provide reliable reference genes for future chordoma research.
Main Methods:
- Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to analyze the expression of 12 candidate reference genes in 18 flash-frozen chordoma samples.
- The stability of candidate genes, including ACTB, B2M, T, EF1a, GAPDH, HPRT, KRT8, KRT19, PGK1, RS27a, TBP, and YWHAZ, was assessed.
- The GeNorm and NormFinder algorithms were utilized to rank gene expression stability.
Main Results:
- GeNorm analysis identified PGK1, YWHAZ, ACTB, HPRT, EF1A, and TBP as the most stable genes.
- NormFinder analysis ranked ACTB, YWHAZ, PGK1, B2M, TBP, and HPRT as the most stable.
- The commonly used reference gene GAPDH demonstrated insufficient stability for reliable results in chordoma gene expression studies.
Conclusions:
- PGK1, ACTB, and YWHAZ emerge as the most stably expressed reference genes in human chordoma samples.
- These genes are recommended as preferred reference genes for gene expression studies in chordoma, surpassing the stability of GAPDH.
- The findings provide essential tools for enhancing the accuracy and reproducibility of chordoma molecular research.
