Primers were designed to amplify both methylated and unmethylated DNA according to the guidelines given in [21 (link)]. ELMO3 primers were: forward; 5′-TGGTTAGGAGTAGTAGTTTGGGT-3′ and reverse; 5′-AATCCTCCCTTTCCGAAACCTA-3′ giving rise to an amplicon of 67 bp. The assay was optimized using a dilution series of methylated DNA (fully methylated from Zymo Research) into unmethylated DNA, prepared as described previously [22 (link)].
PCR cycling and HRM analysis were performed on the Lightcycler® 480 (Roche Applied Science, Mannheim, Germany). The reaction mixtures consisted of 20 ng of bisulfite modified DNA, 0.5 μL of each primer (final concentration of 500 nM), 1.2 μL of MgCl2 (final concentration of 3 mmol/L), 1 μL H20 and 4.8 μL of Lightcycler® 480 High Resolution Melting Master Mix (Roche), in a total volume of 10 μL. The cycling protocol started with one cycle of 95°C for 10 minutes followed by 50 cycles of 95°C for 5 second, 61°C for 10 seconds, 72°C for 10 seconds, one cycle of 95°C for 1 minute, one cycle of 65°C for 1 minute, and a melt from 65°C to 95°C with a temperature increase of 0.1°C/sec. All samples were analyzed in duplicates.
PCR cycling and HRM analysis were performed on the Lightcycler® 480 (Roche Applied Science, Mannheim, Germany). The reaction mixtures consisted of 20 ng of bisulfite modified DNA, 0.5 μL of each primer (final concentration of 500 nM), 1.2 μL of MgCl2 (final concentration of 3 mmol/L), 1 μL H20 and 4.8 μL of Lightcycler® 480 High Resolution Melting Master Mix (Roche), in a total volume of 10 μL. The cycling protocol started with one cycle of 95°C for 10 minutes followed by 50 cycles of 95°C for 5 second, 61°C for 10 seconds, 72°C for 10 seconds, one cycle of 95°C for 1 minute, one cycle of 65°C for 1 minute, and a melt from 65°C to 95°C with a temperature increase of 0.1°C/sec. All samples were analyzed in duplicates.
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