EpiTect MethyLight PCR +ROX Vial Kit (200)

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Publication protocol

MethyLight quantitative polymerase chain reaction (qPCR): The qPCR reactions were performed in 96-well plates on a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), using the EpiTect® MethyLight PCR + ROX Vial kit (Qiagen GmbH; cat. no. 59496) according to the manufacturer's protocol. The reactions were performed in a volume of 20 µl that included: 2X EpiTect MethyLight Master mix (without ROX) 10 µl containing (HotStarTaq Plus DNA Polymerase, EpiTect Probe PCR buffer, deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate and thymidine triphosphate of ultrapure quality); 2 µl 10X primer-probe mix containing 200 nM probe and 400 nM forward and reverse primers; 0.4 µl 50X ROX Dye Solution (passive reference dye for normalization of fluorescent signals); 2 µl (20 ng) bisulfite-treated DNA template; and 5.6 µl RNase-free water. Cycle conditions were as follows: Initial PCR activation step at 95°C for 5 min, 50 cycles at 95°C for 15 sec (denaturation step) and 60°C for 1 min (combined annealing/extension step with fluorescence data collection). Each plate included standard curves for the SPG20 and ALU-C4 sequence, DNA samples, a methylated human bisulfite-converted DNA (EpiTect PCR Control DNA; cat. no. 59655; Qiagen GmbH) as a positive control, unmethylated human bisulfite-converted DNA (EpiTect PCR Control DNA; cat. no. 59665; Qiagen GmbH) as a negative control, unconverted human genomic DNA from healthy human blood samples, which was not amplified, and a no template control, including the entire ingredients of the reaction except the template DNA. The standard curve was plotted using the Cq value (13,14,18,19,34,37) of the methylated human bisulfite-converted DNA at various concentrations vs. the log value of input DNA concentration. An acceptable MethyLight PCR result was obtained, and is demonstrated by the PCR efficiency (96–102%), correct slope value (−3.65 - −3.2) and R2=0.9985. In addition, to establish the standard curve, commercial methylated human bisulfite-converted DNA (EpiTect PCR Control DNA; cat. no. 59655; Qiagen GmbH) was used as a fully methylated control to calculate the percentage of methylated reference (PMR) of the samples. PMR is the degree of methylation of each sample relative to the fully methylated control.

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Manufacturer protocol

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