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Design, measurement and processing of region-specific DNA methylation assays: the mass spectrometry-based method EpiTYPER.

Suchiman HE, Slieker RC, Kremer D, Slagboom PE, Heijmans BT, Tobi EW - Front Genet (2015)

Bottom Line: The technology targets genomic regions of 100-600 base pairs and results in the quantitative measurement of DNA methylation levels largely at single-nucleotide resolution.It is particularly suitable for larger scale efforts to study candidate regions or to validate regions from genome-wide DNA methylation studies.Here, we describe in detail how to design and perform EpiTYPER measurements and preprocess the data, providing details for high quality measurements not provided in the standard EpiTYPER protocol.

View Article: PubMed Central - PubMed

Affiliation: Molecular Epidemiology, Department of Medical Statistics and Bioinformatics, Leiden University Medical Center Leiden, Netherlands.

ABSTRACT
EpiTYPER® is a mass spectrometry-based bisulfite sequencing method that enables region-specific DNA methylation analysis in a quantitative and high-throughput fashion. The technology targets genomic regions of 100-600 base pairs and results in the quantitative measurement of DNA methylation levels largely at single-nucleotide resolution. It is particularly suitable for larger scale efforts to study candidate regions or to validate regions from genome-wide DNA methylation studies. Here, we describe in detail how to design and perform EpiTYPER measurements and preprocess the data, providing details for high quality measurements not provided in the standard EpiTYPER protocol.

No MeSH data available.


Step-down bisulfite PCR.
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Figure 2: Step-down bisulfite PCR.

Mentions: Perform the BS-PCR. Incubate the PCR reactions (5 μl) in standard 384-well plates. Carry out the “step-down” PCR thermal cycling protocol (Figure 2) with 15 min at 95°C, followed by 4 cycles: 20 s at 95°C, 30 s at 65°C, and 1 min at 72°C, then followed by 4 cycles: 20 s at 95°C, 30 s at 58°C, and 1 min at 72°C, and subsequently followed by 38 cycles: 20 s at 95°C, 30 s at [AT]°C* and 1 min at 72°C. Carry out the final extension at 72°C for 3 min, and cool down the sample to 15°C. This cycling protocol takes about 2 h. PCR product can be kept at 4°C. Avoid freeze-thaw cycles.


Design, measurement and processing of region-specific DNA methylation assays: the mass spectrometry-based method EpiTYPER.

Suchiman HE, Slieker RC, Kremer D, Slagboom PE, Heijmans BT, Tobi EW - Front Genet (2015)

Step-down bisulfite PCR.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585020&req=5

Figure 2: Step-down bisulfite PCR.
Mentions: Perform the BS-PCR. Incubate the PCR reactions (5 μl) in standard 384-well plates. Carry out the “step-down” PCR thermal cycling protocol (Figure 2) with 15 min at 95°C, followed by 4 cycles: 20 s at 95°C, 30 s at 65°C, and 1 min at 72°C, then followed by 4 cycles: 20 s at 95°C, 30 s at 58°C, and 1 min at 72°C, and subsequently followed by 38 cycles: 20 s at 95°C, 30 s at [AT]°C* and 1 min at 72°C. Carry out the final extension at 72°C for 3 min, and cool down the sample to 15°C. This cycling protocol takes about 2 h. PCR product can be kept at 4°C. Avoid freeze-thaw cycles.

Bottom Line: The technology targets genomic regions of 100-600 base pairs and results in the quantitative measurement of DNA methylation levels largely at single-nucleotide resolution.It is particularly suitable for larger scale efforts to study candidate regions or to validate regions from genome-wide DNA methylation studies.Here, we describe in detail how to design and perform EpiTYPER measurements and preprocess the data, providing details for high quality measurements not provided in the standard EpiTYPER protocol.

View Article: PubMed Central - PubMed

Affiliation: Molecular Epidemiology, Department of Medical Statistics and Bioinformatics, Leiden University Medical Center Leiden, Netherlands.

ABSTRACT
EpiTYPER® is a mass spectrometry-based bisulfite sequencing method that enables region-specific DNA methylation analysis in a quantitative and high-throughput fashion. The technology targets genomic regions of 100-600 base pairs and results in the quantitative measurement of DNA methylation levels largely at single-nucleotide resolution. It is particularly suitable for larger scale efforts to study candidate regions or to validate regions from genome-wide DNA methylation studies. Here, we describe in detail how to design and perform EpiTYPER measurements and preprocess the data, providing details for high quality measurements not provided in the standard EpiTYPER protocol.

No MeSH data available.