Validation of Reference Genes in Chordoma

Background: In qRT-PCR, reference genes are used for the normalization of gene expression. Ideally, the reference gene should not be regulated or influenced by the experiment. As there is no single reference gene that is stably expressed in all tissues, a validation study is necessary for every tissue type and experimental condition to examine several candidate reference genes. Chordoma are rare tumors of the axial skeleton, which are thought to arise from remnants of the embryonic notochord. So far, no studies have been done to evaluate the stability of reference genes in chordoma. Methods: A total of 13potential reference genes (ACTB, B2M, T, EF1a, GAPDH, HPRT, KRT8, KRT19, PGK1, RPL13a, RS27a, TBP, and YWHAZ) were tested for expression stability on 21 flash frozen chordoma samples with qRT-PCR. GeNorm, NormFinder, and RefFinder algorithms were used to rank the stability of the genes. Results: The four most stable reference genes were: YWHAZ, ACTB, HPRT, and RS27A. According to the GeNorm algorithm, the use of all four genes is recommended. GAPDH alone, which is often used as a reference gene in chordoma, is not stable enough for reliable results. Conclusion: For qPCR analysis, the use of the geometric mean of YWHAZ, ACTB, HPRT, and RS27A as reference is the most optimal setting for a reliable qPCR study.