Ferdowsi University of Mashhad

Document Type : Research Articles

Authors

Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran

Abstract

     Real-time quantitative PCR (qRT-PCR) is often used as an effective experimental method for analyzing gene expression. In this method, normalization of target gene expression levels must be performed using housekeeping genes (HKGs). HKGs are used to compensate for difference between samples due to diverse quality and quality of RNAs and different reverse transcription yield. For an ideal reference gene, constant expression levels across different samples of one experiment is necessary. In the current study, expression stability of four candidate references genes including Beta actin (ACTB), glyceraldeyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyl transferase (HPRT1) and Beta-2-Microglobulin (β2M) following retinoic acid (RA) treatment in embryonal carcinoma NCCIT cells were evaluated.NCCIT cells were exposed to RA (10 µM) for 14 days to induce differentiation. RT-qPCR for candidate references genes was performed and normalization between untreated and RA-treated cells was performed using identical sample input amounts. Expression of OCT4, SOX2, NANOG during RA-induced differentiation was assessed by quantitative real-time PCR. RT-qPCR results indicated significant difference in expression level of GAPDH between untreated (Ct mean: 19.36667± 0.28) and RA-treated (Ct mean: 28.94± 0.18) NCCIT cells. However, transcriptional level of ACTB, HPRT and β2M remained unchanged after RA treatment. qRT-PCR analysis using ACTB, HPRT and β2M showed treatment of NCCIT cells with RA lead to significant down regulation of OCT4 (79%), NANOG (71%) and SOX2 (96%) transcript. ACTB, HPRT and β2M were recognized as valid reference genes for analysis of gene expression during RA-induced differentiation of NCCIT cells, while GAPDH was not suitable.

Keywords

Bustin S. A. (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of molecular endocrinology 25:169-193.
Chambers I. and Tomlinson S. R. (2009) The transcriptional foundation of pluripotency. Development 136:2311-2322.
Chen J., Rider D. A. and Ruan R. (2006) Identification of valid housekeeping genes and antioxidant enzyme gene expression change in the aging rat liver. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:20-27.
de Kok J. B., Roelofs R. W., Giesendorf B. A., Pennings J. L., Waas E. T., Feuth T., Swinkels D. W. and Span P. N. (2005) Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Laboratory investigation 85:154-159.
Dheda K., Huggett J. F., Bustin S. A., Johnson M. A., Rook G. and Zumla A. (2004) Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques 37:112-119.
Glare E. M., Divjak M., Bailey M. and Walters E. H. (2002) β-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels. Thorax 57:765-770.
Guénin S., Mauriat M., Pelloux J., Van Wuytswinkel O., Bellini C. and Gutierrez L. (2009) Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. Journal of experimental botany 60:487-493.
Haller F., Kulle B., Schwager S., Gunawan B., von Heydebreck A., Sültmann H. and Füzesi L. (2004) Equivalence test in quantitative reverse transcription polymerase chain reaction: confirmation of reference genes suitable for normalization. Analytical biochemistry 335:1-9.
Hamalainen H., Tubman J., Vikman S., Kyrölä T., Ylikoski E., Warrington J. and Lahesmaa R. (2001) Identification and validation of endogenous reference genes for expression profiling of T helper cell differentiation by quantitative real-time RT-PCR. Analytical biochemistry 299:63-70.
Janovick-Guretzky N., Dann H., Carlson D., Murphy M., Loor J. J. and Drackley J. K. (2007) Housekeeping gene expression in bovine liver is affected by physiological state, feed intake, and dietary treatment. Journal of Dairy Science 90:2246-2252.
Jin B., Ernst J., Tiedemann R. L., Xu H., Sureshchandra S., Kellis M., and et al. (2012) Linking DNA methyltransferases to epigenetic marks and nucleosome structure genome-wide in human tumor cells. Cell reports 2:1411-1424.
Kooistra S. M., Thummer R. P. and Eggen B. J. (2009) Characterization of human UTF1, a chromatin-associated protein with repressor activity expressed in pluripotent cells. Stem cell research 2:211-218.
Murphy C. L. and Polak J. M. (2002) Differentiating embryonic stem cells: GAPDH, but neither HPRT nor β-tubulin is suitable as an internal standard for measuring RNA levels. Tissue engineering 8:551-559.
Nygard A.-B., Jørgensen C. B., Cirera S. and Fredholm M. (2007) Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC molecular biology 8:67.
Park S.-W., Do H.-J., Ha W. T., Han M.-H., Song H., Uhm S.-J., and et al. (2014) Differential expression of ETS family transcription factors in NCCIT human embryonic carcinoma cells upon retinoic acid-induced differentiation. Biological and Pharmaceutical Bulletin 37:659-665.
Radonić A., Thulke S., Mackay I. M., Landt O., Siegert W. and Nitsche A. (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochemical and biophysical research communications 313:856-862.
Rassouli F. B., Matin M. M., Bahrami A. R., Ghaffarzadegan K., Cheshomi H., Lari S., Memar B. and Kan M. S. (2013) Evaluating stem and cancerous biomarkers in CD15+ CD44+ KYSE30 cells. Tumor Biology 34:2909-2920.
Richly E., Chinnery P. F. and Leister D. (2003) Evolutionary diversification of mitochondrial proteomes: implications for human disease. Trends in Genetics 19:356-362.
Selvey S., Thompson E. W., Matthaei K., Lea R. A., Irving M. and Griffiths L. R. (2001) β-Actin—an unsuitable internal control for RT-PCR. Molecular and cellular probes 15:307-311.
Soltanian S. and Dehghani H. (2018) BORIS: a key regulator of cancer stemness. Cancer cell international 18:1-13.
Soltanian S., Dehghani H., Matin M. M. and Bahrami A. R. (2014) Expression analysis of BORIS during pluripotent, differentiated, cancerous, and non-cancerous cell states. Acta Biochim Biophys Sin 46:647-658.
Soltanian S., Sheikhbahaei M. and Ziasistani M. (2020) Phytol Down-Regulates Expression of Some Cancer Stem Cell Markers and Decreases Side Population Proportion in Human Embryonic Carcinoma NCCIT Cells. Nutrition and Cancer:1-14.
Stevanovic M. (2003) Modulation of SOX2 and SOX3 gene expression during differentiation of human neuronal precursor cell line NTERA2. Molecular biology reports 30:127-132.
Turabelidze A., Guo S. and DiPietro L. A. (2010) Importance of housekeeping gene selection for accurate reverse transcription‐quantitative polymerase chain reaction in a wound healing model. Wound repair and regeneration 18:460-466.
Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A. and et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome biology 3:1-12.
Vossaert L., O’Leary T., Van Neste C., Heindryckx B., Vandesompele J., De Sutter P. and et al. (2013) Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells. BMC molecular biology 14:21.
Wu Y.-Y. and Rees J. L. (2000) Variation in epidermal housekeeping gene expression in different pathological states. Acta dermato-venereologica 80: 2-3
Zhang X., Ding L. and Sandford A. J. (2005) Selection of reference genes for gene expression studies in human neutrophils by real-time PCR. BMC molecular biology 6:4.
Zhong H. and Simons J. W. (1999) Direct comparison of GAPDH, β-actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia. Biochemical and biophysical research communications 259:523-526.
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