Icycler myiq detection system

cfDNA concentrations in plasma were quantified by a direct real-time PCR method (Table 1). For the determination of test and software variations all templates were measured in triplicates. 6.4 µl of diluted plasma were added as template to 41.6 µl master mix containing 1 U/µl Tego Buffer (Bioline, Luckenwalde, Germany), 0.05 U/µl Velocity Polymerase (Bioline, Luckenwalde, Germany), 0.17× SYBR Green (Sigma-Aldrich Co., Taufkirchen, Germany), 0.001 µM FITC (Sigma-Aldrich Co., Taufkirchen, Germany), 0.6 mM MgCl₂ and 0.34 µM each primer. The total volume of reaction mixture per template was 48 µl adequate to three measurements of 15 µl plus pipetting loss. The final plasma volume per well was 0.05 µl. All reactions comprised a triplicate of non-template controls (NTC).
Reactions were carried out in 96-well PCR plates (0.2 ml tube plate, white, Peqlab, Erlangen, Germany) using the iCycler MyIQ Detection System (Biorad, Munich, Germany) for qPCR measurement. Amplification consisted of an initial denaturation for 2 min at 98°C, followed by 35 cycles of melting at 94°C for 10 s, annealing at 64°C for 40 s and extension at 75°C for 10 s. Subsequent qPCR measurements were calibrated to an interplate-calibration template that had been measured several times on plates containing a standard dilution series.

Expression of osteocalcin and gapdh genes was determined by real‐time RT‐PCR using the iCycler MyiQ detection system (Bio‐Rad Laboratories), SYBRGreen (Roche), and specific primers for osteocalcin and gapdh. One microlitre of cDNA was amplified as a template in a 25‐μL reaction mixture containing 12.5 μL 2xSYBRGreen PCR Master Mix (Roche), 2.5 μL of primers and 9 μL deionized water. The mixture was heated initially at 95°C for 5 minutes followed by 40 cycles with denaturation at 95°C for 10 seconds and combined annealing/extension at 60°C for 30 seconds. Data were analysed by the comparative threshold cycle method.