EpiTect Control DNA and Control DNA Set

For control experiments during methylation analysis

Features

  • Convenient, ready-to-use, quality-controlled DNA
  • Bisulfite-converted DNA for control experiments
  • Suitable for all methylation analyses
undefined
EpiTect PCR Control DNA Set (100)

Cat. No. / ID: 59695

Human control DNA set (containing both bisulfite converted methylated and unmethylated DNA and unconverted unmethylated DNA) for 100 control PCRs
EpiTect Control DNA and Control DNA Set are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

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.

Performance

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").
See figures

Principle

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.
See figures

Procedure

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 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

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsEnd point Methylation Specific PCR (MSP), real-time methylation specific PCR
Number of reactionsfor 1000 control PCRs
Concentration10 ng/µl (1 µg in total) excepted the unmethylated control DNA: 50 ng/µl (10 µg in total)

Resources

Kit Handbooks (4)

FAQ

What are the expected PCR results when using EpiTect Control DNA on untreated or bisulfite-converted DNA?

Expected PCR results when using the EpiTect Control DNA Set in combination with the EpiTect MSP Kit can be found in the table below:

 

Type of DNA Primer for unmethylated & unconverted genomic DNA Primer for unmethylated & bisulfite converted target DNA Primer for methylated & bisulfite converted target DNA
Unmethylated & untreated control DNA PCR product NO PCR product NO PCR product
Unmethylated & bisulfite converted control DNA NO PCR product PCR product NO PCR product
Methylated & bisulfite converted control DNA NO PCR product NO PCR product PCR product
No template control NO PCR product NO PCR product NO PCR product

 

 

 

FAQ ID -2011
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
Can the EpiTect PCR Control DNAs be used for mouse and rat assays?

No, the EpiTect PCR Control DNA Set is derived from human DNA. No primers for mouse or rat will bind.

 

 

 

 

 

 

 

 

FAQ ID -2008
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
3341 - How are the EpiTect DNA controls produced and tested?

The genomic DNA is derived from disease free human whole blood from multiple anonymous donors. The DNA is pooled after purification to yield a mixture of individuals (male/female) and the whole genome is represented.   The whole genome is copied stripping the resultant DNA of methylation.

The methylated DNA control is methylated in vitro using SssI methylase. The methylation efficiency is high but not guaranteed to be 100% for every position. Although methylated under optimized conditions for the enzymatic reaction, some positions are as low as 85% as determined by Pyrosequencing.  These sites of lower methylation are not random but rules to predict these low positions are undetermined. 

QIAGEN checks the consistency of EpiTect Control DNA for methylation and bisulfite conversion using an very sensitive real time MSP-PCR assay.  QIAGEN performs several realtime PCR tests to evaluate the bisulfite conversion efficiency as well as the methylation efficiency of the DNAs in the Control DNA Set.  We use test systems specific for genomic DNA to detect unconverted DNA or for converted and unmethylated DNA to detect converted but unmethylated DNA, as well as for converted and methylated DNA.
FAQ - 3341