Blood samples, real-time RT-PCR assay for CK-19 mRNA detection

Five mL peripheral blood was drawn from every patient prior to surgery (t0) and 12 h after the operation (t1) and all laboratory analyses were conducted in a blinded fashion. Mononuclear cells, including tumor cells, were harvested by gradient density centrifugation using Ficoll-Hypaque. Total RNA isolation was carried out by using TRNzol reagent (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. All RNA preparations and handling steps took place in a laminar flow hood under RNAse-free conditions. The isolated RNA was dissolved in diethylpyrocarbonate-treated water and stored at − 80 °C for future use. The purity and quantity of RNA were determined using a spectrophotometer (260 nm/280 nm). Reverse transcription of RNA was carried out with the PrimeScript™ RT reagent (TOYOBO, Japan). Two μg of total RNA was used as template to synthesize cDNA according to the manufacturer’s instructions. The real-time RT-PCR assay for CK-19 mRNA-positive CTCs has been previously described in detail elsewhere (SYBR Green Realtime PCR Master Mix, TOYOBO, Japan). The number of CTCs in each of the tested samples was expressed as Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line equivalents per 2 μg of total-RNA, as determined by LightCycler system (Roche Diagnostics, Germany) software 3.1, according to the external standard calibration curve. According to the analytic detection limit of our assay, the presence of ≥0.9 MCF-7 cell equivalents/2 μg of total RNA was considered a positive result of CTCs, following the protocol of Stathopoulou et al. [17] Using this detecting limit as a cut-off, surgery is considered to implement an increased micrometastatic risk if the number of CTCs is increased beyond this cut-off after operation. Moreover, all blood samples were simultaneously tested for the integrity of RNA by demonstrating positive detection of the house keeping β-actin gene.