Abstract B70: Identification of reference genes for normalizing gene expression for RT-qPCR analysis in established cervical cancer cell lines and their derived cancer stem like cells

Abstract

Abstract Cervical cancer is the fourth most common cancer affecting women worldwide. Cancer stem cells are a subpopulation of tumor cells related to malignancy, resistance, and poor survival. Considering the importance of gene expression studies to understand cervical cancer stem cells (CCSC) biology and the need of proper validation of reference genes to normalize RT-qPCR data, this work identifies stable reference genes for the cervical cancer cell lines SiHa, HeLa, and ME180 as well as their respective derived cancer stem like cells. A literature review was performed to identify whether validation of reference genes was available for RT-qPCR assays in cervical cancer cell lines. Then cell lines were cultured in regular monolayer or in tumor sphere formation conditions. RT-PCR was carried out to validate cells grown as spheres as CCSC. RT-qPCR was performed using five commonly used reference genes: ACTB, B2M, GAPDH, HPRT1, and TBP. Stability was assessed through analysis of dispersion data and the Normfinder software to validate the selected genes as suitable reference genes. According to the literature review, no validation of reference genes for SiHa, HeLa, or ME180 cells in standard culture conditions or their CCSC subpopulations has been done so far. Our evaluation validated B2M, GAPDH, HPRT1, and TBP in these set conditions. Among them, GAPDH presented the lowest stability value (0.222), being therefore the most adequate gene to normalize the combination of all samples. Moreover, associating GAPDH and TBP led to a more stable value (0.184). These results suggest that B2M, GAPDH, HPRT1, and TBP are suitable reference genes to normalize RT-qPCR data of established cervical cancer cell lines SiHa, HeLa, and ME180 as well as their derived cancer stem like cells. Furthermore, GAPDH seems to be the most convenient choice for studying the combination of these cells. Citation Format: Rafael Paschoal de Campos, Iago Carvalho Schultz, Paola de Andrade Mello, Samuel Davies, Manuela Sangalli Gasparin, Ana Paula Santin Bertoni, Jéssica Gonçalves Azevedo, Andréia Buffon, Márcia Rosângela Wink. Identification of reference genes for normalizing gene expression for RT-qPCR analysis in established cervical cancer cell lines and their derived cancer stem like cells [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B70.