EpiTect MethyLight PCR Kits

For quantitative, real-time probe-based PCR analysis of methylation status

Features

  • Highly accurate TaqMan® probe-based real-time analysis
  • Reliable methylation quantification with MethyLight Assays
  • Convenient master mix format
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EpiTect MethyLight PCR +ROX Vial Kit (200)

Cat. No. / ID: 59496

Master Mix without ROX for methylation-specific real-time PCR analysis, 200 x 50 µl reactions
EpiTect MethyLight PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

The EpiTect MethyLight PCR Kit and the EpiTect MethyLight PCR+ROX Vial Kit enable sensitive and reliable quantification for TaqMan® or dual-labeled probe-based methylation analysis. The kits are available in a ready-to-use format, eliminating the need for further optimization. The kits are compatible with a wide range of real-time cyclers. The EpiTect MethyLight PCR Kit is suitable for most cyclers from Applied Biosystems, with the exception of the Applied Biosystems 7500 systems. The EpiTect MethyLight PCR+ROX Vial Kit is suitable for all other cyclers, including the Applied Biosystems 7500 and instruments from Bio-Rad, Cepheid, Corbett, Eppendorf, Roche, and Stratagene.

Performance

The EpiTect MethyLight PCR Kit can sensitively detect even small changes in DNA methylation status (see figure " Sensitive real-time PCR quantification") when used in combination with dual-labeled assays, such as the predesigned EpiTect MethyLight Assays.
See figures

Principle

EpiTect MethyLight PCR Kits can be used in combination with EpiTect MethyLight Assays or other dual-labeled assays for sensitive quantification of DNA methylation in bisulfite converted DNA. Depending on the level used for sequence discrimination, MethyLight analyses can be performed in a quantitative or semiquantitative format. 

Dual-labeled probes, including TaqMan® probes, are sequence-specific oligonucleotides with a fluorophore and a quencher moiety attached. The fluorophore is at the 5' end of the probe, and the quencher moiety is usually located internally or at the 3' end. During the elongation phase of PCR, the probe is cleaved by the 5' → 3' exonuclease activity the polymerase, separating the fluorophore from the quencher moiety. This results in detectable fluorescence that is proportional to the amount of accumulated PCR product.

Quantitative MethyLight real-time PCR with methylation-specific probes

For quantitative methylation analysis using MethyLight PCR, two dual-labeled probes with different fluorescent labels are used in combination with one set of PCR primers. Bisulfite converted DNA requires a different probe sequence for methylated and unmethylated CpG sites. The primers are specific for unmethylated sites. One of the probes binds specifically to methylated DNA at the site of interest, and the other binds specifically to unmethylated DNA at the same site. Since the probes contain different fluorophores, the amounts of methylated and unmethylated sequence can be determined (see figure " Quantitative MethyLight real-time PCR").

Semiquantitative MethyLight real-time PCR with methylation-specific primers

For semiquantitative MethyLight analysis, a dual-labeled probe is used together with a set of PCR primers that specifically binds to the methylation sites of interest. Bisulfite converted DNA requires a different primer sequence for methylated and unmethylated CpG sites, so the primers need to be specific to methylated, bisulfite converted DNA. If the methylation-specific primer binds to the DNA, it will be elongated, and the 5' → 3' exonuclease activity of Taq DNA Polymerase will lead to the degradation of the primer and the release of the fluorophore. As the fluorophore is now separated from the quencher moiety, its fluorescence is detectable (see figure " Semiquantitative MethyLight real-time PCR").

Increased specificity

The EpiTect MethyLight Master Mix has been specifically developed for highly sensitive detection of methylated and unmethylated DNA using sequence-specific probes. In addition to various salts and additives, the buffer also contains a specially optimized combination of KCl and (NH4)2SO4, which promotes a high ratio of specific-to-nonspecific primer and probe binding during the annealing step of each PCR cycle. This allows discriminative hybridization of the probes, and the detection of unmethylated and/or methylated target sequences. The stringent primer annealing conditions lead to increased PCR specificity when amplifying bisulfite converted DNA, enabling reliable probe-based methylation detection.

Control reactions in MethyLight PCR

Control reactions must be carried out to ensure that MethyLight PCR probes and primers specifically bind methylated or unmethylated DNA. Such reactions require bisulfite converted control DNA (fully methylated and fully unmethylated) in various concentrations. In addition, mixtures of these control DNAs can serve as quantification standards when determining methylation degrees. With the EpiTect Control DNA Set, QIAGEN provides the quality-controlled DNAs needed — methylated bisulfite converted, unmethylated bisulfite converted, and normal human genomic DNA — in a ready-to-use kit format for standardized and reliable methylation-specific control reactions.

See figures

Procedure

The EpiTect MethyLight PCR Kit is supplied in a master mix format for a fast and convenient reaction setup. Two kits are available, with and without the inclusion of ROX in the master mix, offering greater flexibility for different real-time cyclers. Validated protocols for most real-time cyclers guarantee optimal results.

The EpiTect MethyLight PCR Kit is supplied with a master mix containing ROX passive reference dye, and is optimized for use with real-time cyclers that require a high concentration of ROX dye for fluorescence normalization (e.g., instruments from Applied Biosystems, excluding the Applied Biosystems 7500 Real-Time PCR Systems).

The EpiTect MethyLight PCR + ROX Vial Kit is supplied with a master mix that does not contain ROX dye, but it includes a separate solution of ROX dye that can be added to reactions, depending on the real-time cycler used. The kit is intended for cyclers that require a lower concentration of ROX dye for fluorescence normalization (e.g., Applied Biosystems 7500 Real-Time PCR Systems), for use with cyclers that allow optional use of ROX dye (e.g., instruments from Stratagene), and for use with cyclers that do not require ROX dye.

Standardized workflows in epigenetics

Accessing epigenetic information is of prime importance for many areas of biological and medical research — particularly oncology, but also stem cell research and developmental biology. However, the analysis of changes in DNA methylation is challenging due to the lack of standardized methods for providing reproducible data particularly from limited sample material. With its newly introduced EpiTect solutions, QIAGEN makes available standardized, pre-analytical and analytical solutions, from DNA sample collection, stabilization and purification, to bisulfite conversion and real-time or end-point PCR methylation analysis or sequencing (see figure " Standardized workflows in epigenetics").

See figures

Applications

EpiTect MethyLight PCR Kits are suitable for quantitative and semiquantitative real-time PCR analysis and can be used in combination with EpiTect MethyLight Assays or other dual-labeled assays.

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsQuantification of bisulfite converted DNA
Reaction typeHighly accurate and sensitive probe-based MethyLight real-time analysis
Real-time or endpointReal-time
With or without ROXAvailable with ROX in master mix and with ROX as separate vial
Sample/target typeBisulfite converted DNA
SYBR Green I or sequence-specific probessequence-specific probes
Single or multiplexSingle & duplex
Thermal cyclerMost real-time cyclers except Applied Biosystems 7500 systems

Resources

Kit Handbooks (1)
EpiTect MethyLight PCR Kit
EpiTect MethyLight PCR + ROX Vial Kit
For quantitative methylation analysis using
sequence-specific probe-based real-time PCR
Brochures & Guides (1)
Now with even more applications!

FAQ

What is the advantage of EpiTect MethyLight PCR Kits over QuantiTect Probe PCR Kits for methylation studies?

The EpiTect MethyLight PCR Kit chemistry is especially developed for the requirements of bisulfite converted DNA. As a result of methylation and the conversion of unmethylated cytosines through bisulfite treatment, probe hybridization behavior differs from that of untreated DNA, and requires special buffer conditions. The balanced cations as well as Factor MP in the MethyLight PCR chemistry are contributing to the special hybridization behavior required for bisulfite converted DNA.

 

 

FAQ ID -2010
Can the EpiTect PCR Control DNAs also be used as standard in real-time methylation specific quantification?

Yes, the EpiTect Control DNA and Control DNA Set can be used as standards in real-time methylation specific quantification. For data, please see our EpiTect MethyLight PCR Product Profile.

 

 

 

FAQ ID -2006
What happens when using EpiTect MethyLight Assays on only partially bisulfite converted DNA template?

If only two of three CpG sites are methylated in the template DNA, EpiTect MethyLight Assays used with the EpiTect MethyLight PCR Kit will detect the site as methylated, irrespective of conversion success for the third CpG site.

However, if only one of three CpG sites is methylated, and the remaining two unmethylated C residues fail to convert to U after bisulfite treatment, the EpiTect MethyLight System will detect the unmethylated site as methylated. Therefore we strongly recommend to use our EpiTect Bisulfite Kits to achieve 100% conversion rate.

 

 

FAQ ID -2009
How long can DNA converted with EpiTect Bisulfite Kits be stored before use in methylation specific PCR with the EpiTect MSP Kit?

We have data for DNA converted with EpiTect Bisulfite Kits showing that the bisulfite treated DNA is stable for 3 years at -20°C before use in methylation specific PCR with the EpiTect MSP Kit, or the EpiTect MethyLight PCR Kit.

 

 

FAQ ID -2002
What is the purpose of the unmethylated and unconverted control DNA of the EpiTect PCR Control DNA Set?

The unmethylated and unconverted human control DNA of the EpiTect PCR Control DNA Set allows to check that primers designed for the specific detection of unmethylated and converted DNA (U-converted DNA), and for methylated, converted DNA (M-converted DNA) do not bind to untreated genomic DNA.*

In case bisulfite conversion was not complete, leaving certain unmethylated C residues unconverted, false positives would result if the primer specific for M-converted DNA binds to untreated gDNA.

This control DNA can also be used to check conversion efficiency during bisulfite treatment.

 

*(Summary of principle: Methylation of DNA occurs on cytosine residues, especially on CpG dinucleotides enriched in small regions of DNA. Incubation of target DNA with sodium bisulfite, using e.g. EpiTect Bisulfite Kits, results in conversion of unmethylated cytosine residues into uracil, leaving methylated cytosines unchanged).

 

FAQ ID -2007