QuantiTect Probe PCR Kit (1000)

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Publication protocol

TaqMan qPCR assays and reaction mix compositions: The TaqMan assays and probe modifications used for the evaluation of the MeltMan system are summarized in Table 1. All the primers and probes used were obtained from Generi-Biotech, Czech Republic, except the minor groove binder (MGB) and locked nucleotide (LNA) modified probes, with the former supplied by Life Technologies and the latter by Roche or Integrated DNA Technologies. Each probe was labelled with a 6-FAM reporter dye and a corresponding quencher. All of the reaction mixes were prepared on the basis of the QuantiTect Probe PCR or QuantiTect Probe RT-PCR Kits (Qiagen), unless stated otherwise. The reactions were set up in a final volume of 25μl (20μl reaction mix and 5μl of NA extract) by using white opaque and foil-sealed plates (LC 480 multiwell plate 96, Roche) or white opaque thin-walled and flat-cap sealed eight-tube strips (Bioplastics). Each of the particular TaqMan assays was set up in two subsets. The first one, called the standard or no S82 subset, contained 0.6μM of primers and 0.2μM of hydrolysis probe. The second one, referred to as the test or S82 subset, contained 0.6μM of primers, 0.4μM of probe and S82 dye (Life Technologies) in a final concentration of 0.8μM. The working solutions of S82 were prepared by dilution in nuclease-free water (Qiagen) and were stored at -20°C with freezing/thaw no more than2-3 times.
Amplification and melting analysis thermoprofiles: The above-mentioned subsets were analysed on the CFX96 (BioRad) thermal cycler, unless stated otherwise. For the QuantiTect Probe PCR Kit, the thermoprofiles universally started with an initial activation at 95°C for 15 min and for the QuantiTect Probe RT-PCR Kit, with a reverse transcription at 50°C for 30 min and 95°C for 15 min. These initial steps were followed by 45 cycles of an assay-specific thermoprofile (Table 1) with a signal acquisition in the FAM and VIC channels at the end of the hybridization (for three-step thermoprofiles) or annealing/extension phase (for two-step thermoprofiles). The amplification was immediately followed by melting analysis ramping from 50°C to 95°C in 0.5°C increments, plate read for 0.5s, and signal acquisition in the VIC channel. For selected reactions the melting analysis was repeated in the LC480 v.1 thermal cycler (Roche) with initial pre-incubation at 40°C for 2 min and a temperature ramp at a rate of 0.06°C/s to 95°C with a continuous signal acquisition mode (10 counts/°C). To read the S82 emission in the LC480v.1 instrument, the following detection format was implemented: excitation and emission filters of 523 and 568, a melt factor of 1.2, a quant factor of 20, and maximum integration time of 1s.
The Cq values were estimated by analyzing the S82 and no S82 data as a single pool using the automatic threshold and baseline cycles option of the CFX Manager Software v3.1. For point 3.7, three additional approaches were implemented i) automatic data analysis separately for the S82 and no S82 reaction subsets, employing the automatic threshold and baseline cycles option; ii) manual threshold adjustment separately for each subsets, and iii) direct Cq inference from the FAM curve trajectories using the qPCR Miner [13] web-based application.
The Cq values were evaluated by constructing the Cq plot and ΔCq scatter plot separately for each of the four baseline approaches used. Within the Cq plot, the no S82 subset Cq values were plotted against the S82 subset values. The ΔCq was calculated according to the formula ΔCq = Cq[no S82]-Cq[S82].

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