A minimal set of internal control genes for gene expression studies in head and neck squamous cell carcinoma

Vinayak Palve,Amritha Suresh,Manisha Pareek,Binay Panda,Moni A Kuriakose,Gangotri Siddappa,Neeraja M Krishnan

doi:10.7717/peerj.5207

Abstract

Selection of the right reference gene(s) is crucial in the analysis and interpretation of gene expression data. The aim of the present study was to discover and validate a minimal set of internal control genes in head and neck tumor studies. We analyzed data from multiple sources (in house whole-genome gene expression microarrays, previously published quantitative real-time PCR (qPCR) data and RNA-seq data from TCGA) to come up with a list of 18 genes (discovery set) that had the lowest variance, a high level of expression across tumors, and their matched normal samples. The 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. Finally, we validated their expression using qPCR in an additional set of tumor:matched normal samples that resulted in five genes (RPL30, RPL27, PSMC5, MTCH1, and OAZ1), out of which RPL30 and RPL27 were most stable and were abundantly expressed across the tissues. Our data suggest that RPL30 or RPL27 in combination with either PSMC5 or MTCH1 or OAZ1 can be used as a minimal set of control genes in head and neck tumor gene expression studies.

Highlights

Internal control genes or housekeeping genes are important in obtaining reliable and reproducible data from gene expression studies in cancer (Eisenberg & Levanon, 2013)
There was some inconsistency between the top-ranked genes among the different algorithms/tools, for the genes RPL27 and RPL30 the data was consistent across all the algorithms
As both of RPL27 and RPL30 belong to the same ribosomal family proteins, and in order to avoid any potential error due to mutual expression alterations of those genes, we considered inclusion of the three genes PSMC5, MTCH1, and OAZ1, the expression of which were stable across all the samples

Summary

Introduction

Internal control genes or housekeeping genes are important in obtaining reliable and reproducible data from gene expression studies in cancer (Eisenberg & Levanon, 2013). Internal control genes should be abundantly and uniformly expressed across tumor and normal tissues and across different experimental conditions (Janssens et al, 2004). Past studies argue the lack of uniformity of expression on internal control genes based on experimental conditions (De Jonge et al, 2007; Greer et al, 2010). There are previous reports that describe the reference or internal control genes in head and neck squamous cell carcinoma. How to cite this article Palve et al (2018), A minimal set of internal control genes for gene expression studies in head and neck squamous cell carcinoma.

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