4.7. Quantitative PCR (Q-PCR)
cancer, mammary tumors
PLACENTA, CANCER, MAMMARY TUMORS
This study is a subset of the clinical trial. Fifteen consecutive women in each group receiving CEE/MPA and EGene expression levels were quantified by Q-PCR using Taqman Gene Expression Assays and Taqman Gene Expression Master Mix (Life Technologies) in multiplex reactions. mRNA levels were normalized to the level of endogenous control 18S. Sixteen probes for target genes, eleven genes identified as increasing mammary tumors by the IPA database, and an additional five genes with specific relevance for hormonal effects/risk of cancer in mammary tissue were assessed (We ran the reactions in a Step One 7300 Real-Time multiplex PCR system (Life Technologies). All the reactions contained 10 ng of cDNA in a 25 µL reaction volume. The PCR efficiency with all amplicons was 90–100%, and we performed all determinations in triplicates and included duplicate negative (no-template) and positive controls (Human Placenta Total RNA, Lot No. 030302520J, Ambion Life Technologies, Austin, TX, USA).Q-PCR reactions were performed on 30 consecutive patients (15 from each treatment group) both at baseline and at the end of the treatment in 96-well optical PCR plates. Target gene TaqMan probes were FAM™ dye-labeled, and 18S cDNA probes were VIC™ dye-labeled; all products, including oligonucleotide primers, were purchased from Applied Biosystems. All plates included 18S rRNA amplification of each sample as an endogenous control for data normalization. The cycling conditions were: 50 °C for 2 min, followed by 95 °C for 10 min, then 40 cycles of 95 °C for 15 s and 60 °C for 1 min. For all sixteen genes, we used the same PCR primers as for the previous microarray.We analyzed the Q-PCR data using the comparative Ct method, where Ct is the cycle number when the fluorescence first exceeds the threshold, and calculated ΔCt by subtracting the Ct value of the endogenous control from the Ct value of the target gene. This quantification gave us the RQ value [