Computational protocol: 18FDG-PET/CT and molecular markers to predict response to neoadjuvant chemotherapy and outcome in HER2-negative advanced luminal breast cancers patients

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Protocol publication

[…] Quantitative PCR analysis was performed on 10 ng cDNA in duplicate. A 5 μL diluted sample of cDNA was added to 20 μL of the PCR mix. The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min, 45 cycles at 95°C for 15 sec, and annealing temperature, either 60°C or 65°C depending on the target, for 1 min.All PCR reactions were performed using the ABI Prism 7500 Sequence Detection System (Applied Biosystems Inc., Forster City, USA). The PCR Core reagent kit was used for systems with Taqman probes (Eurogentec, Liège, Belgium). Primers and fluorescent probes were designed from published sequences using Primer express software (Applied Biosystems Inc.). BLASTN searches against dbEST and nr (the non-redundant set of the GenBank sequence database) were performed to confirm the total gene specificity of the chosen nucleotide sequences and the absence of DNA polymorphisms. Primers and probes sequences for Ki67, and MYBL2, KPNA2, CDC2, CDC20 mRNA expression, as components of the GGI, are presented in of supplementary data. TATA Box binding protein (TBP) was used as endogenous reference genes. Target quantities were normalized to TBP mRNA expression.Human breast luminal cancer cell lines T47D cDNA was used to generate 7 points standard curves for each gene. Target quantities were normalized to the reference genes and calibrated using the second point of each standard curve. Final results were expressed as N-fold differences in target gene expression relative to the reference genes and the calibrator and are expressed as:2target(Ct calibrator - Ct sample)/Ereference gene(Ct calibrator - Ct sample),where Ct is the cycle threshold.No reverse-Transcription Controls (NTC) were included in any batch of samples. […]

Pipeline specifications

Software tools Primer Express, BLASTN
Databases dbEST
Application qPCR
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms