Reference gene for primary culture of prostate cancer cells

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 specific to knockdown the androgen receptor and cells transfected with inespecific 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, flavoprotein (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 find 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.

Effect of Helicobacter pylori on NFKB1, p38α and TNF-α mRNA expression levels in human gastric mucosa

“…These studies determined that SDHA was the best reference gene compared with other frequently used reference genes (27,60,61). According to the results of the present and previous studies, SDHA may be used as a reference gene for qPCR in the conditions described, as a result of its stability.…”

“…To select a reference gene, five reference genes commonly used in gene expression studies were examined to identify the most appropriate gene for the current experiment. The primers were supplied by Laboratory of Molecular Biology, Endocrinology and Tumors (Federal University of Rio Grande do Sul, Porto Alegre, Brazil) (27). Each primer was synthesized by Invitrogen Brazil, Ltd. (São Paulo, Brazil).…”

“…In order to perform gene expression analysis, the suitability of five reference genes were assessed to determine their suitability for use in qPCR normalization. Although the five genes have been widely studied (27,28), there are presently no studies evaluating their association with H. pylori infection and human gastric inflammation.…”

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“…Four reference genes -TBP, HPRT1, ALAS1 and TUBA1B -were used for nor maliza tion of RE levels [4,7] . RE levels were calculated, using two common methods (2 -ΔCt and 2 -ΔΔCt ) described earlier [10][11][12].…”

“…The validation of the reference genes for prostate tumors, lymph nodes from patients with prostate cancer and also prostate cancer cell lines resulted in the creation of a set of genes, namely TBP, HPRT1, ALAS1, TUBA1B, GAPDH and B2M that are expressed constitutively in prostate cancer and normal tissues, making them suitable for qPCR normalization [6][7][8][9].…”