Primers used for sequencing are displayed in Additional file 2 Table S2. PCR products were purified by using ExoSAP-IT (USB, Cleveland, USA) and DNA sequencing reactions were performed in both directions using BigDyeTerminator v3.1 (Applied Biosystems, Selleckchem CA4P Nieuwerkerk a/d IJssel, the Netherlands)
on a 48-capillary 3730 DNA Analyzer sequencer (Applied Biosystems, Nieuwerkerk a/d IJssel, the Netherlands). Accession numbers: HQ222846 to HQ222861 and HQ606074. PCR and real-time qPCR Oligonucleotides were synthesized by Biolegio (Biolegio, Nijmegen, the Netherlands). Conventional PCR was used to produce amplicons from signature sequences. Amplification was carried out using the HotStar Taq Master Mix Kit (Qiagen, Westburg, the Netherlands) and 400 nM primers in a total reaction volume of 50 μl. Primer sets were designed using Visual OMP software (Additional file 2 Table S2). Thermocycling conditions Temsirolimus mouse were as follows: 95°C for 15 min, 40 cycles at 95°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec, followed by a final step at 72°C for 7 min.
Thermocycling reactions were carried out in a Px2 thermal cycler (Thermo Electron Corporation, Breda, the Netherlands). All qPCR reactions were carried out in a final volume of 20 μl containing iQ Multiplex Powermix (Bio-Rad, Veenendaal, the Netherlands), 200 nM of each primer and 100-300 nM hydrolysis probes and 3 μl of DNA template. Probes concentrations had been optimized to yield minimal spectral overlap between fluorescence level of the reporter dyes for each target in a multiplex assay and were 100, 200, 300 and 300 nM for FAM, JOE, CFR590 and Cy5 labeled probes respectively. The multiplex real-time qPCR selleck inhibitor assays had been designed for an optimal annealing temperature of 60°C and the thermal cycling conditions were as follows: First enzyme activation at 95°C for 5 min, followed by amplification and detection by 45 cycles at 95°C for 5 sec and 60°C for 35 sec. Each real-time
qPCR experiment included a negative (no template) control. Measurements were carried out on a Lightcycler 480 3-mercaptopyruvate sulfurtransferase (Roche, Almere, the Netherlands). An iQ5 (Bio-Rad) instrument was used for routine screening purposes. Analyses were performed on the instruments software: LightCycler 480 Software release 1.5.0. SP3 and iQ5 Optical Systems Software version 2.0. Cq values were calculated using the second derivative method on the LightCycler and the Base Line Subtracted Curve Fit method on the iQ5. Color compensations were carried out on both instruments as follows. PCR amplifications were performed using single primer-probe sets for each reporter dye and under identical reaction conditions as during multiplex amplification. The PCR reactions thus produced contained single dyes in relevant concentrations and these were used for color compensation runs according to the manufacturers’ guidelines.