Identification of reference genes for qRT-PCR in human lung squamous-cell carcinoma by RNA-Seq

Abstract

Although the accuracy of quantitative real-time polymerase chain reaction (qRT-PCR) is highly dependent on the reliable reference genes, many commonly used reference genes are not stably expressed and as such are not suitable for quantification and normalization of qRT-PCR data. The aim of this study was to identify novel reliable reference genes in lung squamous-cell carcinoma. We used RNA sequencing (RNA-Seq) to survey the whole genome expression in 5 lung normal samples and 44 lung squamous-cell carcinoma samples. We evaluated the expression profiles of 15 commonly used reference genes and identified five additional candidate reference genes. To validate the RNA-Seq dataset, we used qRT-PCR to verify the expression levels of these 20 genes in a separate set of 100 pairs of normal lung tissue and lung squamous-cell carcinoma samples, and then analyzed these results using geNorm and NormFinder. With respect to 14 of the 15 common reference genes (B2M, GAPDH, GUSB, HMBS, HPRT1, IPO8, PGK1, POLR2A, PPIA, RPLP0, TBP, TFRC, UBC, and YWHAZ), the expression levels were either too low to be easily detected, or exhibited a high degree of variability either between lung normal and squamous-cell carcinoma samples, or even among samples of the same tissue type. In contrast, 1 of the 15 common reference genes (ACTB) and the 5 additional candidate reference genes (EEF1A1, FAU, RPS9, RPS11, and RPS14) were stably and constitutively expressed at high levels in all the samples tested. ACTB, EEF1A1, FAU, RPS9, RPS11, and RPS14 are ideal reference genes for qRT-PCR analysis of lung squamous-cell carcinoma, while 14 commonly used qRT-PCR reference genes are less appropriate in this context.

Tissue Morphology and Gene Expression Characterisation of Transplantable Adenocarcinoma Bearing Mice Exposed to Fluorodeoxyglucose-Conjugated Magnetic Nanoparticles

“…39 43 44 In one study, PGK1 and PPIA (Peptidyl-prolyl cis-trans isomerase A) were undetectable by RNA-Seq in human lung squamous cell carcinoma, and so were deemed inappropriate for use as reference genes. 45 The differences between these observations and ours may be explained by the analogous comparison to the difference between human and mouse samples, as well as the difference between analyses in cell line and primary tissues. Indeed, several studies have shown that the selection of the most appropriate reference gene in cancer tissue is dependent on the specific nature of the experiment.…”

Variation in transcriptome size: are we getting the message?

“…In RT-PCR or qRT-PCR, the expression of a target gene is normalized to the expression of a putatively stably expressed reference gene (Vandesompele et al 2002). In practice, the stability of expression for a potential reference gene is assessed by comparison to other genes (Vandesompele et al 2002) or based on expression profiles from microarrays (Libault et al 2008;Cheng et al 2011) or RNA-Seq (Zhan et al 2014). In other words, reference genes are selected whose expression is assumed to comprise a constant fraction of the transcriptome.…”

Identification of long noncoding RNAs for the detection of early stage lung squamous cell carcinoma by microarray analysis

“…Each sample was measured in triplicate and a negative control was included with every RT-qPCR assay. β-actin was used as the reference gene instead of GAPDH since a recent study reported that the latter had differential expression between LSCC and NT samples [42]. Relative expression levels were quantified based on the crossing point values and normalized to the reference gene.…”