Abstract
BackgroundThe selection of reference genes is essential for quantifying gene expression. Theoretically they should be expressed stably and not regulated by experimental or pathological conditions. However, identification and validation of reference genes for human cancer research are still being regarded as a critical point, because cancerous tissues often represent genetic instability and heterogeneity. Recent pan-cancer studies have demonstrated the importance of the appropriate selection of reference genes for use as internal controls for the normalization of gene expression; however, no stably expressed, consensus reference genes valid for a range of different human cancers have yet been identified.ResultsIn the present study, we used large-scale cancer gene expression datasets from The Cancer Genome Atlas (TCGA) database, which contains 10,028 (9,364 cancerous and 664 normal) samples from 32 different cancer types, to confirm that the expression of the most commonly used reference genes is not consistent across a range of cancer types. Furthermore, we identified 38 novel candidate reference genes for the normalization of gene expression, independent of cancer type. These genes were found to be highly expressed and highly connected to relevant gene networks, and to be enriched in transcription-translation regulation processes. The expression stability of the newly identified reference genes across 29 cancerous and matched normal tissues were validated via quantitative reverse transcription PCR (RT-qPCR).ConclusionsWe reveal that most commonly used reference genes in current cancer studies cannot be appropriate to serve as representative control genes for quantifying cancer-related gene expression levels, and propose in this study three potential reference genes (HNRNPL, PCBP1, and RER1) to be the most stably expressed across various cancerous and normal human tissues.
Highlights
The selection of reference genes is essential for quantifying gene expression
Given that Transcripts per million (TPM) is established to produce more comparable results across various sample types than Reads per kilobase per million mapped reads (RPKM) [13, 14], we used TPM-generated data for 32 of the 34 cancer types for further analyses [esophageal carcinoma (ESCA) and stomach adenocarcinoma (STAD) were excluded, since only RPKM-generated data were available for these cancer types]
Used reference genes exhibit a high level of expression variation in both tumorous and normal tissue samples To assess the gene expression variability within human cancerous and normal tissues, we collected gene expression data from the The Cancer Genome Atlas (TCGA) database, which contains 10,028 (9,364 cancerous and 664 normal) samples isolated from 32 different cancer types
Summary
The selection of reference genes is essential for quantifying gene expression. Theoretically they should be expressed stably and not regulated by experimental or pathological conditions. Recent pan-cancer studies have demonstrated the importance of the appropriate selection of reference genes for use as internal controls for the normalization of gene expression; no stably expressed, consensus reference genes valid for a range of different human cancers have yet been identified. An increasing number of researches have shown the striking expression variability of known reference genes in human cancers, and subsequently recommended novel reference genes for gene expression studies in each specific human cancer type [6, 7] These efforts with in silico analysis (e.g., geNorm, NormFinder, and Bestkeeper [8,9,10]) are ongoing; to date, no transcriptome-wide analysis for the identification of the most stably expressed consensus reference genes has been reported
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
