EZ DNA Methylation-Direct Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Upstream tips
Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Publication protocol

Methylation specific PCR (MSP)
DNA bisulfite modification and purification were performed using an EZ DNA methylation-Direct kit (Zymo Research Corporation, Irvine, California, USA) according to the instruction. Concentration of DNA was evaluated by absorbance at 260 nm on a UV spectrophotometer (BioRad Inc., Hercules, CA, USA). The sets of primers for miR-30a/c-5p gene were flanking the 3kb regions of the 5’ upstream regions from the start of pre-miR-30a/c-5p sequences. The primers for methylation-specific PCR were designed by MethPrimer and the sequences were as follows: miR-30a methylated (M)-forward (F): 5’-TAAATATTTTAAGGATAGAGAGCGA-3’, miR-30a methylated (M)-reverse (R): 5’-AAAATTTAAAAACAACCCGAA-3’, miR-30a unmethylated (U)- forward (F): 5’-AAATATTTTAAGGATAGAGAGTGA-3’, miR-30a unmethylated (U)- reverse (R): 5’-ATAAAAAATTTAAAAACAACCCAAA-3’; miR-30c methylated (M)-forward (F): 5’-AGGCGTTTGTAGTTTTAGTTATTCG-3’, miR-30c methylated (M)-reverse (R): 5’-CGAAATAAAATCTCGCTCTATCG-3’, miR-30c unmethylated (U)- forward (F): 5’-GTGTTTGTAGTTTTAGTTATTTGG-3’, miR-30c unmethylated (U)- reverse (R): 5’-TATACTCCAACTACTCACACCACG-3. DNA amplification was performed with Epi Taq HS (Takara Biotechnology Co. Ltd., Dalian, China) under the following condition: 94 °C for 5 min; 40 cycles of 94 °C for 45 s, 44.3 °C (miR-30a U)/48 °C (miR-30a M)/36 °C (miR-30c U)/50 °C (miR-30c M)/for 45 s, 72 °C for 1 min; and 72 °C for 10 min. The PCR products were separated by 2.0 % agarose gel electrophoresis and visualized by a chemiluminescence imaging system (Bio-Rad, Richmond, CA, USA).

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Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing DNA methylation profiling Gene specific profiling - A2780 miR-30c-5p using EZ DNA Methylation-Direct Kit from Zymo Research.

Manufacturer protocol

Download the product protocol from Zymo Research for EZ DNA Methylation-Direct Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing DNA methylation profiling Gene specific profiling - A2780 miR-30c-5p using EZ DNA Methylation-Direct Kit from Zymo Research. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.