A minimal set of internal control genes for gene expression studies in head and neck squamous cell carcinoma
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Abstract
Background
Selection of the right reference gene(s) is crucial in the analysis and interpretation of gene expression data. In head and neck cancer, studies evaluating the efficacy of internal reference genes are rare. Here, we present data for a minimal set of candidates as internal control genes for gene expression studies in head and neck cancer.
Methods
We analyzed data from multiple sources ( in house whole-genome gene expression microarrays, n =21; TCGA RNA-seq, n =42, and published gene expression studies in head and neck tumors from literature) to come up with a set of genes (discovery set) for their stable expression across tumor and normal tissues. We then performed independent validation of their expression using qPCR in 14 tumor:normal pairs. Genes in the discovery set were ranked using four different algorithms (BestKeeper, geNorm, NormFinder, and comparative delta Ct) and a web-based comparative tool, RefFinder, for their stability and variance in expression across tissues.
Results
Our analyses resulted in 18 genes (discovery set) that had lowest variance and high level of expression across tumor and normal samples. Independent experimental validation and analyses with multiple tools resulted in top ranked five genes ( RPL30, RPL27, PSMC5, OAZ1 and MTCH1 ) out of which, RPL30 (60S ribosomal protein L30) and RPL27 (60S ribosomal protein L27), performed best and were abundantly expressed across tumor and normal tissues.
Conclusions
RPL30 and RPL27 are stably expressed in HNSCC and should be used as internal control genes in gene expression in head and neck tumors studies.
