Abstract
BackgroundThe selection of suitable internal control genes is crucial for proper interpretation of real-time PCR data. Here we outline a strategy to identify housekeeping genes that could serve as suitable internal control for comparative analyses of gene expression data in breast cancer cell lines and tissues obtained by high throughput sequencing and quantitative real-time PCR (qRT-PCR).MethodsThe strategy proposed includes the large-scale screening of potential candidate reference genes from RNA-seq data as well as their validation by qRT-PCR, and careful examination of reference data from the International Cancer Genome Consortium, The Cancer Genome Atlas and Gene Expression Omnibus repositories.ResultsThe identified set of reference genes, also called novel housekeeping genes that includes CCSER2, SYMPK, ANKRD17 and PUM1, proved to be less variable and thus potentially more accurate for research and clinical analyses of breast cell lines and tissue samples compared to the traditional housekeeping genes used to this end.DiscussionThese results highlight the importance of a massive evaluation of housekeeping genes for their relevance as internal control for optimized intra- and inter-assay comparison of gene expression.ConclusionWe developed a strategy to identify and evaluate the significance of housekeeping genes as internal control for the intra- and inter-assay comparison of gene expression in breast cancer that could be applied to other tumor types and diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2946-1) contains supplementary material, which is available to authorized users.
Highlights
The selection of suitable internal control genes is crucial for proper interpretation of real-time PCR data
We described a strategy for the selection of protein targets suitable for drug development against neoplastic diseases taking the case of breast cancer (BC) as a pertinent example [14]
We propose a strategy to identify potential novel housekeeping genes (HKG) and to validate traditional HKGs (tHKGs) that may serve as internal controls in BC investigations based on high throughput sequencing (HTS) and quantitative real-time PCR (qRT-PCR)
Summary
The selection of suitable internal control genes is crucial for proper interpretation of real-time PCR data. We outline a strategy to identify housekeeping genes that could serve as suitable internal control for comparative analyses of gene expression data in breast cancer cell lines and tissues obtained by high throughput sequencing and quantitative real-time PCR (qRT-PCR). High-throughput technologies allow genome-wide expression profiling, which is already providing important insights into complex regulatory networks, enabling the identification of new or under-explored biological processes, and helping to uncover the genes that are involved in various pathological processes as is the case with cancer [2, 3]. Because of Statistical consistency involves experimental reproducibility and, from a general viewpoint, reproducibility is an absolute prerequisite for reliable inference, especially when investigating the biological significance of subtle differences in gene expression [9]. Experimental reproducibility is generally linked to the concept of robustness that is understood as the stability of a system output (here, the gene expression) with respect to stochastic
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