Gentra Puregene Cell Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Frozen samples should be thawed and equilibrated to room temperature (15–25°C) before beginning the procedure.

Upstream tips
Frozen samples should be thawed and equilibrated to room temperature (15–25°C) before beginning the procedure.

Publication protocol

2.6. DNA extraction and qPCR analysis of promoter methylation
Genomic DNA was extracted using Qiagen Puregene Core Kit A according to the manufacturer's protocol and quantified using NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). CpG island DNA methylation of individual genes was analyzed using the EpiTect Methyl qPCR Assay (Qiagen). The method, referred as “qAMP”, is based on the detection of DNA remaining after cleavage with methylation-sensitive and/or methylation-dependent restriction enzymes. This method has been already reported to provide accurate and reproducible results without the requirement for sodium bisulfite treatment of DNA [12].

After digestion of unmethylated and methylated DNAs by methylation-sensitive and methylation-dependent restriction enzymes, residual DNA was quantified by qPCR using primers flanking the promoter region of interest. The relative fractions of hypermethylated (HM) and unmethylated (UM) and intermediately methylated (IM) DNAs were subsequently determined by comparing the amount in each digest with a control untreated sample. The reaction mix was prepared according to manufacturer's instructions: briefly, four 50 ng of genomic DNA samples were digested at 37 °C for 16 h either with methylation-sensitive, methylation-dependent, mock or both restriction enzymes. Reactions were blocked by heat-inactivation at 65 °C for 20 min and PCR performed applying the following thermal protocol: 95 °C for 10 min; 3 cycles at 99 °C for 30 s and 72° for 1 min; 40 cycles at 97 °C for 15 s and 72 °C for 1 min. Ct values were used to calculate the percentage of HM, UM and IM DNAs using the datasheet provided at http://www.sabiosciences.com/dna_methylation_data_analysis1.php (Individual EpiTect Methyl qPCR Assay).

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Manufacturer protocol

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