Validation of reference genes in human chordoma

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.