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EpiTect MethyLight PCR Kits

For quantitative, real-time probe-based PCR analysis of methylation status
  • Highly accurate TaqMan® probe-based real-time analysis
  • Reliable methylation quantification with MethyLight Assays
  • Convenient master mix format
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.
Cat No./ID: 59436
EpiTect MethyLight PCR Kit (200)
€816.00
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Master Mix for methylation-specific real-time PCR analysis, 200 x 50 µl reactions
Cat No./ID: 59496
EpiTect MethyLight PCR +ROX Vial Kit (200)
€816.00
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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.


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0
Sensitive real-time PCR quantification.
EpiTect MethyLight Assays enable the direct quantification of the degree of methylation in a sample by using the threshold cycle values (CT) determined by qPCR. [A] In the FAM channel, the probe detects methylated DNA, and in the [B] VIC channel, the probe detects unmethylated DNA. A 10 µg sample of bisulfite converted, methylated and unmethylated human control DNA, or defined mixtures of both DNAs containing 95% to 5% methylated DNA, as indicated in the figure, were used in a methylation quantification experiment on the ABI PRISM 7900 cycler. The EpiTect MethyLight Assay for HS_CDKN2A was used in combination with the EpiTect MethyLight PCR Kit. The degree of methylation was calculated from the CT values from the FAM (CT (CG)) and VIC (CT (TG)) channels.
1
Quantitative MethyLight real-time PCR.
[A] and [C] In methylation-specific TaqMan® assays, two methylation-sensitive TaqMan® probes are present together with methylation-insensitive PCR primers. Depending on the methylation status of the targeted sequence, only the TaqMan® probe specific for bisulfite converted methylated DNA or the TaqMan® probe specific for unmethylated DNA can hybridize to the target sequence. [B] and [D] Both probes are labeled with different fluorophores that are released during PCR if the probe hybridizes to the DNA. The fluorescence from the released fluorophore is proportional to the amount of accumulated PCR product.
2
Semiquantitative MethyLight real-time PCR.
[A] and [C] In semiquantitative MethyLight PCR, a TaqMan® probe is located between primers that bind the methylation sites of interest. Primers are specific for methylated sequences, annealing only to methylated DNA, and not to unmethylated DNA. During the PCR extension step, the fluorophore is released, and the resulting fluorescence is measured. [B] and [D] In the case of unmethylated DNA, primer annealing and extension does not occur, and no fluorescence is detected. Ideally, a separate assay is carried out with primers specific for converted, unmethylated DNA, to determine the amount of unmethylated sequences. To quantify the methylation degree, a separate assay determining the DNA quantity is required (not shown in the figure).
3
Standardized workflows in epigenetics.
  
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.
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.

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").

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.

Features
Specifications
Applications Quantification of bisulfite converted DNA
Reaction type Highly accurate and sensitive probe-based MethyLight real-time analysis
Real-time or endpoint Real-time
Sample/target type Bisulfite converted DNA
Single or multiplex Single & duplex
SYBR Green I or sequence-specific probes sequence-specific probes
Thermal cycler Most real-time cyclers except Applied Biosystems 7500 systems
With or without ROX Available with ROX in master mix and with ROX as separate vial
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