Validation of reference genes for the normalization of RT-qPCR expression studies on human laryngeal cancer and hypopharyngeal cancer.

Abstract

Selecting stably expressed reference genes is crucial for evaluating real-time quantitative polymerase chain reaction (RT-qPCR) data via the relative quantification method. In the present-day study, our aim was to select optimal reference genes (RGs) for the investigation of target gene (TG) expression profiling in cancerous human laryngeal and hypopharyngeal tissues. 12 cancerous laryngeal tissues and 10 cancerous hypopharyngeal tissues were investigated. The expression characteristics of 11 reference genes (18S rRNA, GAPDH, B2M, ACTB, TBP, ALAS1, RPL29, HMBS, HPRT1, GUSB, and PUM1), which were commonly used in RT-qPCR for the analysis of gene expression, were investigated using the geNorm, NormFinder, and BestKeeper algorithm programs. HMBS, ALAS1, and B2M were suggested as optimal RGs for studying human laryngeal and hypopharyngeal cancerous tissues together, laryngeal cancerous tissue by itself, and hypopharyngeal cancerous tissue by itself, respectively. If 2 or more reference genes are needed to achieve better standardization, 3 reference genes can optimally be used in combination to improve the accuracy of relative quantitation normalization. The recommended combinations for studying human laryngeal and hypopharyngeal cancerous tissues together, laryngeal cancerous tissue by itself, and hypopharyngeal cancerous tissue by itself were HMBS + HPRT1 + GUSB, ALAS1 + GUSB + HMBS, and B2M + HPRT1 + TBP, respectively. The recommended reference genes could be used to improve the accuracy of gene expression studies on the molecular mechanisms of cancerous human laryngeal and hypopharyngeal tissues. The selected combination of reference genes can effectively improve the accuracy of the relative quantitative diagnosis of gene expression levels, such as messenger RNA, circular RNA, and long-noncoding RNA.