Magmedip qpcr kit

The DNA methylation of the cells from the cell cultures was analyzed using MagMeDIP qPCR Kit (Diagenode, Denville, NJ, USA) according to the manufacturer’s protocol. After precipitation and DNA extraction, DNA concentration was measured, using Pico488 dsDNA quantification reagent (Lumiprobe, Cockeysville, MD, USA). The standard curve was prepared from human DNA quantified by Quantifiler™ Duo DNA Quantification Kit (Thermo Fisher, Waltham, MA, USA). The global DNA methylation was quantified as the percentage of DNA immunoprecipitated using C-me antibodies (Diagenode, Denville, NJ, USA) to the input amount of DNA, based on the concentration results. The site-specific DNA methylation was analyzed in Real-Time PCR, using Fast SYBR Green Master Mix (Thermo Fisher, Waltham, MA, USA). The primer sequences are presented in Table 1. The promoter region of the IL10 gene was the target of primers. The calculation of % of the input was done according to the manufacturer’s protocol. The following formula was used:
where, CtInput—Ct value of 10% Input; CtCme—Ct value of precipitated DNA using Cme antibody.

DNA methylation was analyzed using the MagMeDIP qPCR Kit (Diagenode, Denville, NJ, USA) according to protocol. After precipitation and DNA extraction, the DNA concentration was measured using Pico488 dsDNA quantification reagent (Lumiprobe, Hannover, Germany). The standard curve was prepared using human DNA quantified by the Quantifiler™ Duo DNA Quantification Kit (ThermoFisher Scientific). Global DNA methylation was calculated as the percentage of DNA immunoprecipitated using C^me antibodies (Diagenode) to the input amount of DNA. The site-specific DNA methylation was analyzed in real-time PCR using the Fast SYBR Green Master Mix (ThermoFisher Scientific). The primer sequences are presented in Table 1. The calculation of percent of input was performed according to the manufacturer protocol:
CtIN—Ct value of 10% input; CtIP—Ct value of immunoprecipitated samples].

MeDIP was performed to detect immunoprecipitated methylated DNA with an anti-5′-methyl-cytosine antibody. The MagMeDIP qPCR kit (Diagenode, Belgium) was used following manufacturer’s instructions. Briefly, genomic DNA was sheared using a BioRuptor sonicator (Diagenode, Belgium) to produce 400 bp fragments, which were checked by gel electrophoresis. The obtained fragments were immunocaptured with a monoclonal antibody specific for 5-methyl cytosine (supplied by the kit). Methylated DNA was then washed and purified from beads with a DNA Isolation Buffer (DIB) and proteinase K provided by the kit. Following an incubation at 55°C and at 100°C (for 15 min each), the supernatant containing the DNA was used for qPCR analysis to evaluate enrichment.