Maxima hot start taq pcr buffer

qPCR reactions were performed in a total volume of 20 μl with a volume of cDNA corresponding to 20 or 25 ng of purified RNA, 9.5 pmol primer pair, 4 nmol dNTPs (dATP, dCTP, dTTP, dGTP), 45 nmol MgCl₂, 10% [v/v] Maxima Hot Start TaqPCR buffer, 0.5 U Maxima Hot Start TaqDNA Polymerase (all Thermo Scientific), 1% [v/v] ROX reference dye and 8% [v/v] of SYBR Green Nucleic Acid Stain diluted 1:10000 (both from Life Technologies). Reactions were performed with a 7500 Fast Real-time PCR System in sealed 96-well optical reaction plates (both from Life Technologies) for 40 cycles (15 s at 90 °C, 60 s at 60 °C) after 10 min denaturation at 95 °C. Amplicon sizes were evaluated by melting-curve analysis (95 °C, 15 s, 60 °C 60 s, 95 °C, 30 s, 60 °C 15 s). Relative expression of target genes was determined by normalization to the respective housekeeping genes. Ct-values were determined automatically by the 7500 Software v2.0.5. (Relative expression of target genes was calculated by determination of 2^−ΔΔCt-values.) Used primer pairs (all from Sigma-Aldrich) are listed in supplemental Table S4.

Bisulfite-modified DNA was used as a template for MSP. Two sets of primers covering the promoter region of the MT1E gene (positioned at -112/+105 and -239/-91 from transcription start site, MT1E-1 and MT1E-2, respectively) were designed with Methyl Primer Express software v1.0 (Applied Biosystems) and ordered from Metabion (Martinsried, Germany). The reaction mix (25 μL) contained 1x Maxima Hot Start Taq PCR buffer, 2.5 mM MgCl₂, 1.6 mM dNTP mix, 1.25 U Maxima Hot Start Taq DNA polymerase (Thermo Fisher Scientific, Vilnius, Lithuania), 1 μM of each primer, and 1 μL of bisulfite-modified DNA. MSP reaction conditions included 37-38 cycles with primer annealing step at 55-56°C for 45 s. Bisulfite-modified leukocyte DNA from healthy donors served as a negative control for methylated DNA and SssI methyltransferase-treated (Thermo Fisher Scientific, Vilnius, Lithuania) bisulfite-modified leukocyte DNA served as a positive control. Non-template controls were included in each MSP run.

DNA was extracted from fresh-frozen tissue samples following the standard phenol-chloroform purification protocol as described previously [10 (link)]. Four hundred ng of purified DNA were bisulfite-modified, using the EZ DNA Methylation™ Kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s instructions, and analyzed by methylation-specific PCR (MSP). MSP primers, overlapping with the location of the microarray probes of interest, were designed with Methyl Primer Express Software v1.0 (Applied Biosystems™, Thermo Fisher Scientific, Carlsbad, CA, USA) and ordered from Metabion (Martinsried, Germany) (Table S2). The reaction mix (25 μL) consisted of 1× Maxima Hot Start Taq PCR buffer, 2.5 mM MgCl₂, 0.4 mM of each dNTP, 1.25 U Maxima Hot Start Taq DNA Polymerase (all from Thermo Scientific™, Thermo Fisher Scientific, Vilnius, Lithuania), 1 µM of each primer, and 10–20 ng of bisulfite-treated DNA. Reaction conditions were optimized before the study and included 35–38 cycles with primer annealing step 55–62 °C for 45 s (Table S2). Methylation-positive (MC), methylation-negative, and non-template controls (NTC) were included in each MSP assay.