Epitect pcr control dna kit

Bisulfite conversion was performed using the EpiTect Plus DNA Bisulfite Kit (Qiagen) and 40 μL of the isolated cfDNA, according to the manufacturer’s instructions. Bisulfite-converted cfDNA was then eluted in 30 μL of elution buffer.
For the PCR amplification of the LINE-1 repetitive region using the PyroMark PCR Kit (Qiagen) 1 μL of bisulfite-treated DNA was used as the template. Samples along with methylated and unmethylated control DNA from the EpiTect PCR Control DNA kit (Qiagen) were run in triplicates. PCR protocol was as follows: initial denaturation at 95°C for 15 min; 50 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s; final extension was at 72°C for 10 min (Daskalos et al., 2009 (link)). The biotinylated PCR product was purified using the Pyromark Q24 Vacuum Workstation (Qiagen). Methylation levels of the six CpG’s were then measured by Pyromark Q24 Advanced System with PyroMark Q24 CpG Advanced Reagents (Qiagen). cfDNA methylation levels were calculated as the ratio of C/T at a CpG site using the Pyromark Q24 Advanced Software 3.0.1 (Qiagen). Global cfDNA methylation was calculated as the average value of the six CpG’s. Primers used in cfDNA methylation analysis are listed in Table 4.

After determination of the DNA content using Quantus photometer and QuantiFluor dsDNA-System Kit (Promega, Mannheim, Germany), 80 ng of the purified DNA fragments from unmethylated genomic control DNA (EpiTect PCR Control DNA kit, Qiagen) after pre-amplification were bisulfite modified using EpiTect Fast DNA Bisulfite Kit (Qiagen GmbH) according to manufacturer's instructions. The resulting bisulfite modified fragments were eluted in 40 μl EB buffer and quantified with 9.72 × 10⁸ copies/μl (3.0 × 10⁸ copies/ng) of unmethylated PLA2R1 DNA fragments using ddPCR. Correspondingly, DNA fragments isolated from U937 cells and bisulfite modified methylated PLA2R1 were quantified with 6,500 copies/μl. After determination of the cfDNA content isolated from pooled serum samples ranging between 152 ng and 2,200 ng (Table 2) all the isolated cfDNA were bisulfite modified and the bisulfite-converted samples were eluted in 45 μl EB buffer and stored at – 80°C until analysis.

Genomic DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). DNA was bisulfite converted using the EpiTect Kit (QIAGEN, Hilden, Germany) according to
the manufacturer’s instructions. MSP was carried out using SFRP2 promoter methylation and non-methylation-specific primer pairs (table 2) designed in previous studies. ^{21 (link)}MSP was carried out for each sample in a final reaction volume of 20 µL, containing 0.4 µM of primers, 10 µL of Taq DNA Polymerase Master Mix RED (10 μL)
(Ampliqon, Stenhuggervej, Denmark), and 50 ng of bisulfite-treated DNA.
MSP was performed on the Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Polymerase chain reaction (PCR) conditions were as follows:
initial denaturation for three minutes at 94 ºC, followed by 40 cycles of 30 seconds at 94 ºC, 30 seconds at 64 ºC, 30 seconds at 72 ºC, and final elongation for seven minutes
at 72 ºC. Unmethylated and completely methylated DNAs, contained in the EpiTect PCR Control DNA Kit (QIAGEN, Hilden, Germany), were used as positive controls.
Electrophoresis on a 2.5% agarose gel was done for the identification of the MSP product.