Rotor gene q real time pcr platform

A BRAF RGQ PCR Kit (Qiagen, Hilden, Germany) was used according to the manufacturer's instructions. This assay is designed to detect a wild-type control and the four most common BRAF mutations V600E/K/R/D. Real-time PCR was performed on a Rotor-Gene Q Real-time PCR Platform (Qiagen, Hilden, Germany). The cycling conditions for quality control runs and mutation assays were as follows: 15 min at 95°C, followed by 40 cycles at 95°C for 30 s and 60°C for 1 min. Fluorescence was measured at 60°C. Data on each mutation were interpreted according to the kit manual after a curve analysis and calculation of ΔCt values.

Reverse transcription reactions were performed using SuperScript III Reverse Transcriptase (Invitrogen, Grand Island, NY, USA) with approximately 5 μg of total RNA, following the manufacturer's instructions. Primers for qPCR were designed using Primer Premier 5 software (listed in additional file 2 in Supplementary Material available online at http://dx.doi.org/10.1155/2014/381501).
β-Actin was used as the internal control gene. qPCR was performed on a Qiagen Rotor-Gene Q real-time PCR platform (Qiagen, Hilden, Germany) using SYBR-Green real-time PCR mix (Transgene) to detect the gene expression level. The amplification was performed as follows: initial denaturation at 95°C for 30 s, followed by 45 cycles of denaturation at 95°C for 5 s, and annealing and extension at 55°C for 30 s. The relative expression levels of the genes were normalized toβ-actin and calculated using the 2^−ΔΔCt method. All reactions were performed with three replicates. Statistical analysis was performed using the Pearson correlation test in the SPSS19.0 software. A P value < 0.05 was considered statistically significant.