The selection of stably expressed reference genes is crucial for quantitative real-time polymerase chain reaction (qRT-PCR) assay when assessing osteosarcoma treated with cyclophosphamide (CTX) and Ginkgo biloba L. extract (EGB). We aimed to identify the reference genes that are stably expressed in human osteosarcoma cell lines after the above treatments. | Turkish Journal of Biology Turk J Biol (2017) 41: 439-447 © TÜBİTAK doi: Research Article Reference gene expression in human osteosarcoma cell lines treated by EGB and CTX 1, 2, 3 1 1,2 Ming REN *, Qiwei YANG *, Yuanyuan SONG , Ao WANG , Qingyu WANG , 1 2 1 1,2 1,2 1, Xiaonan WANG , Shuhong HAO , Zhitao WANG , Zhenwu DU , Guizhen ZHANG , Jincheng WANG ** 1 Department of Orthopedics, Second Hospital, Jilin University, Changchun, Jilin, . China 2 Central Laboratory, Second Hospital, Jilin University, Changchun, Jilin, . China 3 Department of Epidemiology and Biostatistics, School of Public Health, Jilin University, Changchun, Jilin, . China Received: Accepted/Published Online: Final Version: Abstract: The selection of stably expressed reference genes is crucial for quantitative real-time polymerase chain reaction (qRT-PCR) assay when assessing osteosarcoma treated with cyclophosphamide (CTX) and Ginkgo biloba L. extract (EGB). We aimed to identify the reference genes that are stably expressed in human osteosarcoma cell lines after the above treatments. We used qRT-PCR assay to determine the expression levels of 18S, ACTB, ALAS1, GAPDH, TBP, HPRT1, RPL29, HMBS, PPIA, PUM1, GUSB, and B2M in the cell lines MG-63, Saos-2, and U2OS, which were treated with CTX or EGB or both. We analyzed the expression stability of these genes using geNorm, NormFinder, and BestKeeper. The optimal reference gene combinations were 18S + PPIA + HPRT1 in the total group, HMBS + ALAS1 + 18S in the EGB-treated group, PPIA + RPL29 in the EGB + CTX-treated group, PPIA + HPRT1 in the MG-63 group, 18S + PPIA in the Saos-2 group, and HPRT1 + ALAS1 + GAPDH in the U2OS group. We conclude that no single reference gene was most stably expressed in all three treated cell lines. Our findings provide a suitable approach through which qRT-PCR can be applied to investigate the pharmacological .
