For control experiments during methylation analysis
Convenient, ready-to-use, quality-controlled DNA
Bisulfite-converted DNA for control experiments
Suitable for all methylation analyses
EpiTect Control DNAs are ready-to-use, completely methylated or completely unmethylated bisulfite converted DNAs, and untreated, unmethylated genomic DNA, for standardized and reliable control reactions for methylation analysis. The methylated, bisulfite converted DNA as well as unmethylated, bisulfite converted and unconverted DNA are stored in EB Buffer (10 mM Tris Cl) in a ready-to-use solution.
Human control DNA set (containing both bisulfite converted methylated and unmethylated DNA and unconverted unmethylated DNA) for 100 control PCRs
Real-time PCR standards.
EpiTect Methylated Control DNA and EpiTect Unmethylated Control DNA (both pre-bisulfite converted) were mixed to give 100%, 90%, 50%, 10%, and 0% methylated DNA. EpiTect MethyLight Assays for the human PITX2 gene were run in triplicate, using 10 ng of each DNA sample. The results show that EpiTect Control DNA can be used as a methylation standard for the quantification of unknown DNA samples.
HRM quantification standards.
A CpG island from the promoter region of the APC gene (adenomatosis polyposis coli) was amplified, and the degree of methylation was determined by HRM methylation analysis on the Rotor-Gene Q, using the EpiTect HRM PCR Kit. Methylated and unmethylated DNA from the EpiTect Control DNA Set was mixed in varying ratios, and used as the templates for the analysis.
EpiTect Control DNA Pyrogram.
Sixteen CpGs of the MGMT promoter were checked for complete methylation (mean methylation rate >95%) or complete nonmethylation (mean methylation rate 0.3%). The PCR products for MGMT (6-O methylguanine-DNA methyltransferase), which is a p53-related gene involved in DNA repair and drug resistance, were subjected to Pyrosequencing, which was performed on a PyroMark Q96ID instrument. (Data kindly provided by Uwe Gerstenmaier, Varionostic GmbH, Ulm).
EpiTYPER MALDI-TOF analysis of WBA DNA.
Methylation of the MP6 locus was measured in unmethylated DNA (UMDNA 1), methylated DNA (VIAL A 1), and a mixture of both (HTXA 1), before and after amplification (WBAUMDNA1, WBAHTXA 1, WBAVIAL A 1) using the EpiTect Whole Bisulfitome Kit. The methylation pattern prior to amplification is very similar to that of the amplified DNA, demonstrating representative amplification. (Data kindly provided by Hany Ezzeldin, Mayo Clinic, Rochester, USA).
EpiTect Control DNAs are highly suited for use as quantification standards for probe-based real-time PCR methylation analysis (see figure "Real-time PCR standards") and high-resolution melting (HRM) methylation analysis (see figure "HRM quantification standards").
The EpiTect Control DNA Set contains all control DNAs needed for standardized and reliable methylation control reactions in a ready-to-use kit format . In methylation-specific PCR, for example, control reactions must be carried out to ensure that PCR probes and primers bind specifically to methylated or unmethylated DNA (see figure Use of EpiTect Control DNA in PCR for methylation analysis). These reactions require various concentrations of completely bisulfite converted control DNA that is fully methylated or fully unmethylated (see figures EpiTect Control DNA Pyrogram and "EpiTYPER MALDI-TOF analysis of WBA DNA"). Additionally, mixtures of these control DNAs can serve as quantification standards when determining degree of methylation in HRM and probe-based methylation experiments.
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").
EpiTect Control DNAs are suitable for use in control reactions for all types of methylation analyses, including:
Evaluating primer specificity for MSP
Quantification standard for HRM and MethyLight PCR
Evaluating primer and probe specificity for MethyLight PCR
Checking efficiency of bisulfite conversion reactions
End point Methylation Specific PCR (MSP), real-time methylation specific PCR
10 ng/µl (1 µg in total) excepted the unmethylated control DNA: 50 ng/µl (10 µg in total)
For 200 reactions: 2x PyroMark PCR Master Mix (includes HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer containing 3 mM MgCl2 and dNTPs), 10x CoralLoad Concentrate, 5x Q-Solution, 25 mM MgCl2, and RNase-Free Water
For 100 x 50 µl reactions: QIAGEN OneStep RT-PCR Enzyme Mix (1 x 200 µl), 5x QIAGEN OneStep RT-PCR Buffer (1 x 1 ml), dNTP Mix (1 x 200 µl, 10 mM each), 5x Q-Solution (1 x 2 ml), RNase-Free Water (2 x 1.9 ml)
For 800 reactions: 2x PyroMark PCR Master Mix (includes HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer containing 3 mM MgCl2 and dNTPs), 10x CoralLoad Concentrate, 5x Q-Solution, 25 mM MgCl2, and RNase-Free Water