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Mol Biol Rep (2013) 40:29552962 DOI 10.1007/s11033-012-2366-5
Reference gene for primary culture of prostate cancer cells
Aline Francielle Damo Souza Ilma Simoni Brum
Brasil Silva Neto Milton Berger Gisele Branchini
Received: 12 July 2012 / Accepted: 17 December 2012 / Published online: 27 December 2012 Springer Science+Business Media Dordrecht 2012
Abstract Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specic to knockdown the androgen receptor and cells transfected with inespecic siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in
this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, avoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to nd the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs.
Keywords Prostate cancer cells Primary culture
Housekeeping genes siRNA
Introduction
Prostate cancer (PCa) is the sixth most common cancer in the world and the most prevalent in men, accounting for 10 % of all types of cancers. In Brazil PCa is the second main cause of cancer death among men [1]. Despite the rising knowledge about the hormonal, nutritional, and environmental context of PCa, several mechanisms concerning the pathogenesis of prostate cancer have to be...