MethylEasy™ Xceed (ME002)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.

Protocol tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.

Publication protocol

Fresh pituitary glands and livers were removed in 20 mM HEPES buffer under a stereomicroscope (Leica S8AP0, Leica Microsystems, Newcastle, UK). Genomic DNA was extracted using a NucleoSpin Tissue XS (MACHEREY-NAGEL, Düren, Germany) from pooled embryonic (sex unknown) and postnatal male tissues as follows: pituitary gland on E13.5 (n = 10), E14.5 (n = 8), P4 (n = 5), and P30 (n = 2); and liver on E13.5 (n = 2), P4 (n = 5), and P30 (n = 2), respectively. Bisulfite conversion of genomic DNA was performed using a MethylEasy Xceed kit (Human Genetic Signatures Pty, North Ryde, Australia) according to manufacturer instructions. Following bisulfite conversion, genomic DNA was amplified by PCR using the BIOTAQ DNA polymerase (Bioline Reagents, London, UK) with primer sets specific for bisulfite-converted genomic DNA sequences (Table 1). PCR conditions were as follows: 94°C for 10 min, 12 cycles of 94°C for 30 sec, and 66°C for 30 sec, with a decrease of 0.5°C each cycle, and 72°C for 60 sec; or 40 cycles of 94°C for 30 sec, 58°C to 62°C for 30 sec, and 72°C for 60 sec; either condition was followed by a final extension of 72°C for 10 min. Amplicons were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA), and positive clones were confirmed by PCR, followed by DNA sequencing using a BigDye Terminator version 2.0 system (Applied Biosystems, Waltham, MA, USA) and an ABI3130 sequencer (Applied Biosystems). DNA sequencing and methylation data were visualized using QUMA software (http://quma.cdb.riken.jp/).

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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 - Rat whole pituitary glands PROP1 using MethylEasy™ Xceed (ME002) from Genetic signatures.

Manufacturer protocol

Download the product protocol from Genetic signatures for MethylEasy™ Xceed (ME002) below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing DNA methylation profiling Gene specific profiling - Rat whole pituitary glands PROP1 using MethylEasy™ Xceed (ME002) from Genetic signatures. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.