GATA4 Methylation Analysis via MSP

GATA4 gene promoter methylation was analyzed using methylation-specific PCR (MSP). The isolated genomic DNA was modified with sodium bisulfide using the EpiTect Bisulfite Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s guidelines. Converted and purified DNA was stored at −80 °C before use. As a positive and negative control, EpiTect methylated/unmethylated human control DNA (bisulfite converted) (Qiagen, Valencia, CA, USA) were used. The reaction mixture (20 μL volume) contained 50–100 ng of bisulfite-treated DNA as a template, 0.5 U TaKaRaEpiTaq HS (TaKaRaClontech Laboratories, Inc., San Jose, CA, USA), 2.5 mM MgCl2, 0.3 mM dNTP Mixture, 1× EpiTaq PCR Buffer, and 0.3 µM of the primers. The amplification was performed in Labcycler 48 Gradient (SensoQuest Bio-medical electronics, Göttingen, Germany). The primer sequences were as follows: forward methylated-5′-GTATAGTTTCGTAGTTTGCGTTTAGC-3′; reverse methylated-5′-AACTCGCGACTCGAATCCCCG-3′ (product of 136 bp); forward unmethylated 5′- TTTGTATAGTTTTGTAGTTTGTGTTTAGT-3′; and reverse unmethylated 5′-CCCAACTCACAACTCAAATCCCCA-3′ (product of 142 bp).
The conditions for the GATA4-methylated amplicons were: 95 °C for 15 min, 38 cycles 95 °C for 30 s, 65 °C for 1 min, 72 °C for 1 min, and 72 °C for 7 min; in addition, for the GATA4-unmethylated amplicons, the conditions were: 95 °C for 15 min, 38 cycles 95 °C for 30 s, 62 °C for 1 min, 72 °C for 1 min, and 72 °C for 7 min. Each PCR reaction included a positive control (methylated DNA), negative control (unmethylated DNA), and water. Each PCR product (6 µL) was directly loaded onto 2% agarose gel and visualized under a UV illuminator (Syngen G: BOX). Methylation-specific PCR for GATA4 was performed on 23 samples due to the unavailability of the material. Representative MSP results are shown in Figure 1.