MethylEasy™ Xceed (ME002)

DNA methylation profiling Gene specific profiling - Rat whole pituitary glands PROP1

Experiment
DNA methylation profiling Gene specific profiling - Rat whole pituitary glands PROP1
Product
MethylEasy™ Xceed (ME002) from Genetic signatures
Manufacturer
Genetic signatures

Protocol tips

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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Discussion

Discussion

5 years ago

Author: Jody Hancock Canada

How can I preserve leukocytes for DNA methylation profiling?

I would like to preserve leukocytes for future epigenetic analysis. How can I preserve them effectively in order to perform DNA methylation profiling at a later time?

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Papers

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Manufacturer protocol

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

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Videos

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