Validation of reference genes in human chordoma

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.