Identification and Validation of Reference Genes for RT-qPCR Studies of Hypoxia in Squamous Cervical Cancer Patients
Open Access
- 31 May 2016
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 11 (5), e0156259
- https://doi.org/10.1371/journal.pone.0156259
Abstract
Hypoxia is an adverse factor in cervical cancer, and hypoxia-related gene expression could be a powerful biomarker for identifying the aggressive hypoxic tumors. Reverse transcription quantitative PCR (RT-qPCR) is a valuable method for gene expression studies, but suitable reference genes for data normalization that are independent of hypoxia status and clinical parameters of cervical tumors are lacking. In the present work, we aimed to identify reference genes for RT-qPCR studies of hypoxia in squamous cervical cancer. From 422 candidate reference genes selected from the literature, we used Illumina array-based expression profiles to identify 182 genes not affected by hypoxia in cervical cancer, i.e. genes regulated by hypoxia in eight cervical cancer cell lines or correlating with the hypoxia-associated dynamic contrast-enhanced magnetic resonance imaging parameter ABrix in 42 patients, were excluded. Among the 182 genes, nine candidates (CHCHD1, GNB2L1, IPO8, LASP1, RPL27A, RPS12, SOD1, SRSF9, TMBIM6) that were not associated with tumor volume, stage, lymph node involvement or disease progression in array data of 150 patients, were selected for further testing by RT-qPCR. geNorm and NormFinder analyses of RT-qPCR data of 74 patients identified CHCHD1, SRSF9 and TMBIM6 as the optimal set of reference genes, with stable expression both overall and across patient subgroups with different hypoxia status (ABrix) and clinical parameters. The suitability of the three reference genes were validated in studies of the hypoxia-induced genes DDIT3, ERO1A, and STC2. After normalization, the RT-qPCR data of these genes showed a significant correlation with Illumina expression (PBrix (PSTC2 data were associated with clinical outcome, in accordance with the Illumina data. Thus, CHCHD1, SRSF9 and TMBIM6 seem to be suitable reference genes for studying hypoxia-related gene expression in squamous cervical cancer samples by RT-qPCR. Moreover, STC2 is a promising prognostic hypoxia biomarker in cervical cancer.Keywords
Funding Information
- The Research Council of Norway (226120/O30)
This publication has 48 references indexed in Scilit:
- Increased expression of stanniocalcin 2 is associated with tumor progression after radiotherapy in patients with cervical carcinoma.2014
- Quantifying mRNA and MicroRNA with qPCR in Cervical Carcinogenesis: A Validation of Reference Genes to Ensure Accurate DataPLOS ONE, 2014
- Selection of Reliable Reference Genes for RT-qPCR AnalysisPublished by Springer Science and Business Media LLC ,2014
- Gene expression analysis in prostate cancer: The importance of the endogenous controlThe Prostate, 2012
- STC2: A Predictive Marker for Lymph Node Metastasis in Esophageal Squamous-Cell CarcinomaAnnals of Surgical Oncology, 2010
- Stanniocalcin 2 overexpression in castration‐resistant prostate cancer and aggressive prostate cancerCancer Science, 2009
- Selecting control genes for RT-QPCR using public microarray dataBMC Bioinformatics, 2009
- Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancerBMC Molecular Biology, 2007
- Tumor Hypoxia Has Independent Predictor Impact Only in Patients With Node-Negative Cervix CancerJournal of Clinical Oncology, 2002
- Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genesGenome Biology, 2002