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Journal of Molecular and Clinical Medicine   2018, Vol. 1 Issue (3): 127-134    DOI: 10.31083/j.jmcm.2018.03.001
Research article | Next articles
Identification of appropriate housekeeping genes for quantitative RT-PCR analysis in MDA-MB-231 and NCI-H460 human cancer cell lines under hypoxia and serum deprivation
Amanda P. B. Albuquerque1, 2, Meritxell Balmaña3, 4, Celso A. Reis3, 4, 5, 6, *(), Eduardo I.C. Beltrão1, 2, *()
1 Biomarkers in Cancer Research Group (BmC) - Federal University of Pernambuco (UFPE), 50670-901 Recife, Brazil
2 Department of Biochemistry- Federal University of Pernambuco (UFPE), 50670-420 Recife, Brazil
3 Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
4 Institute of Molecular Pathology and Immunology, University of Porto, 4200-135 Porto, Portugal
5 Institute of Biomedical Sciences Abel Salazar, University of Porto, 4050-313 Porto, Portugal
6 Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal
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Gene expression studies aimed at analyzing cancer cells under hypoxia and serum deprivation conditions show major potential for understanding molecular mechanisms associated with tumor progression as well as resistance to antitumor agents. To the best of our knowledge, a study for the identification of appropriate housekeeping genes in breast and lung cancer cells under hypoxia and serum deprivation conditions is currently missing. Given the relevance of a reliable and accurate normalization, we herein aimed to identify the appropriate housekeeping genes for breast and lung cancer cell lines cultured under hypoxia and/or serum deprivation. The stability of five commonly used housekeeping genes (ACTB, β2M, GUSB, 18S rRNA, and PPIA) was assessed after reverse-transcription quantitative real-time PCR in MDA-MB-231 and NCI-H460 cancer cell lines using GeNorm, NormFinder and BestKeeper software. GeNorm and NormFinder ranking revealed ACTB, GUSB and PPIA as the most stable genes for both tumor cell lines. Our results support the use of ACTB/PPIA for MDA-MB-231 and GUSB/PPIA for NCI-H460 cells as the most stable combination for normalization of gene expression under hypoxic and serum deprivation conditions. Our results highlight the importance of the selection of the housekeeping genes in cancer cells subjected to different physiological stresses, such as hypoxia and serum deprivation.

Key words:  Breast cancer      Housekeeping genes      Hypoxia      Lung cancer      RT-qPCR      Serum deprivation     
Submitted:  29 March 2018      Revised:  29 May 2018      Accepted:  06 July 2018      Published:  20 September 2018     
*Corresponding Author(s):  Celso A. Reis,Eduardo I.C. Beltrão     E-mail:;

Cite this article: 

Amanda P. B. Albuquerque, Meritxell Balmaña, Celso A. Reis, Eduardo I.C. Beltrão. Identification of appropriate housekeeping genes for quantitative RT-PCR analysis in MDA-MB-231 and NCI-H460 human cancer cell lines under hypoxia and serum deprivation. Journal of Molecular and Clinical Medicine , 2018, 1(3): 127-134.

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Table 1  Primer sequences used for the expression analysis of housekeeping genes
Symbol Gene name Primer sequences (5→3’) Product size/bp R2 E(%)
ACTB Actin, beta F: agaaaatctggcaccacacc R: tagcacagcctggatagcaa 173 0.99 90
β2M Beta-2-microglobulin F: agcgtactccaaagattcaggtt R: atgatgctgcttacatgtctcgat 206 1 105
GUSB Beta-glucuronidase F: agccagttcctcatcaatgg R: ggtagtggctggtacggaaa 160 1 90
PPIA Peptidylprolyl isomerase A F: agacaaggtcccaaagac R: accaccctgacacataaa 118 1 100
18S 18S ribosomal F: cgccgctagaggtgaaattc R: cattcttggcaaatgctttcg 67 0.99 90
Fig. 1.  Mean Ct values of HKG candidates. (A) MDA-MB-231 and (B) NCI-H460 cells cultured in normoxia with FBS supplementation (21% O$_{2}$, 10% FBS$_{ }$-N10), hypoxia with FBS supplementation (1% O$_{2}$, 10% FBS$_{ }$-H10) and normoxia and hypoxia without FBS supplementation, N0 and H0, respectively, for 48 h. Data are expressed as the mean $\pm $ standard deviation. Ct, threshold cycle.

Fig. 2.  Expression stability values of HKG candidates analyzed by GeNorm, NormFinder and BestKeeper softwares. GeNorm average expression stability measures (M) of HKG candidates in MDA-MB-231 (A) and NCI-H460 (B) cell lines. NormFinder ranking of HKG candidates and their expression stability in MDA-MB-231 (C) and NCI-H460 (D) cell lines. Correlation coefficient ($r)$ values of the HKG candidates analyzed by BestKeeper software in MDA-MB-231 (E) and NCI-H460 (F) cell lines.

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