Identification of Suitable Housekeeping Genes for Quantitative Gene Expression Analysis During Retinoic Acid-induced Differentiation of Embryonal Carcinoma NCCIT Cells

     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-۳-phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyl transferase (HPRT۱) and Beta-۲-Microglobulin (β۲M) following retinoic acid (RA) treatment in embryonal carcinoma NCCIT cells were evaluated.NCCIT cells were exposed to RA (۱۰ µM) for ۱۴ 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 OCT۴, SOX۲, 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: ۱۹.۳۶۶۶۷± ۰.۲۸) and RA-treated (Ct mean: ۲۸.۹۴± ۰.۱۸) NCCIT cells. However, transcriptional level of ACTB, HPRT and β۲M remained unchanged after RA treatment. qRT-PCR analysis using ACTB, HPRT and β۲M showed treatment of NCCIT cells with RA lead to significant down regulation of OCT۴ (۷۹%), NANOG (۷۱%) and SOX۲ (۹۶%) transcript. ACTB, HPRT and β۲M were recognized as valid reference genes for analysis of gene expression during RA-induced differentiation of NCCIT cells, while GAPDH was not suitable.

Keywords:

Housekeeping genes , NCCIT cell , Retinoic acid , Differentiation , Expression analysis