EpiTect MSP Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.	Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Upstream tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.
Protocol tips
Always include fully methylated (in-vitro methylated DNA) and non-methylated controls (Leucocyte DNA, DNMT1/3 double KO DNA) during MSP

Publication protocol

DNA from human tissues was extracted from 20 EOC and 20 normal ovarian tissue samples using the QIAmp DNA Mini kit (Qiagen). Bisulfite treatment was performed with the EpiTect Bisulfite kit (Qiagen) in accordance with the manufacturer’s instructions. The methylation status of the MEG3 gene was determined using conventional MSP. For the MSP, we used the EpiTect MSP kit (Qiagen). The sequences of the methylation-specific primers were as follows: the methylated pair (M), 5′-GTT AGT AAT CGG GTT TGT CGG C (forward) and 5′-AAT CAT AAC TCC GAA CAC CCG CG (reverse); the unmethylated pair (U), 5′-GAG GAT GGT TAG TTA TTG GGG T (forward) and 5′-CCA CCA TAA CCA ACA CCC TAT AAT CAC A (reverse). MEG3 DNA was amplified in a DNA Engine Peltier thermal cycler (Bio-Rad) by 1 cycle of 95°C for 15 min followed by 5 cycles of 94°C for 30 sec, 70°C for 30 sec and 72°C for 30 sec; 5 cycles of 94°C for 30 sec, 65°C for 30 sec and 72°C for 30 sec; 30 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec; and 1 cycle of 72°C for 7 min. The PCR products were identified by electrophoresis through a 2.5% agarose gel and ethidium bromide staining (12).

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing DNA methylation profiling Gene specific profiling - Human ovarian tissue MEG3 using EpiTect MSP Kit from Qiagen.

Manufacturer protocol

Download the product protocol from Qiagen for EpiTect MSP Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing DNA methylation profiling Gene specific profiling - Human ovarian tissue MEG3 using EpiTect MSP Kit from Qiagen. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.