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PyroMark CpG Assays

Predesigned assays for quantification of CpG methylation by Pyrosequencing


  • Advanced assay design algorithms provide optimized results
  • Easy-to-order assays for gene-specific or methylation array CpG sites
  • Comprehensive coverage of CpG sites in human, mouse, and rat genomes
  • Convenient download of run files and analysis settings
  • Top performance from a cost-effective solution
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Find or custom design the right target-specific assays and panels to research your biological targets of interest.
PyroMark CpG Assay 96 well (200)

Cat. No. / ID: 978756

PCR and sequencing primers for Pyrosequencing analysis of gene-specific CpG methylation after DNA bisulfite conversion (200 reactions; plate format); minimum order of 24 assays per plate
Configure at GeneGlobe   to see pricing.
PyroMark CpG Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
Configure at GeneGlobe
Find or custom design the right target-specific assays and panels to research your biological targets of interest.

Product Details

PyroMark CpG Assays are the only genomewide, predesigned methylation assays for Pyrosequencing analysis that use a tailored design algorithm for highly specific assay design and successful CpG methylation results. Our upgraded assay database now includes over 84,000 individual assays for gene-specific human, mouse, and rat CpG sites. We now also offer predesigned assays for validating results from methylation arrays, such as the Infinium HumanMethylation450K and MethylationEPIC BeadChip arrays.


Primer design optimized for high performance
Highly specific and unbiased amplification is especially important for methylation analysis by Pyrosequencing. Because bisulfite converted DNA is a less complex template, it can be challenging to amplify specifically and at high yields, but with predesigned PyroMark CpG Assays, this is not a problem. The PCR and sequencing primers included in PyroMark CpG Assays have been checked against the entire human, mouse, or rat bisulfitome and are delivered at optimal concentrations for high performance. Together with the unique DNA protect technology of EpiTect Fast Bisulfite Conversion Kits and the carefully formulated reagents of the PyroMark PCR Kit, these predesigned assays ensure high yield and quality of sequencing template and increase reliability of downstream Pyrosequencing analyses.

Evaluation of PCR performance for 310 randomly selected PyroMark CpG Assays showed that 305 assays (98%) produced the predicted PCR amplicon, suitable for use as Pyrosequencing template. From these assays, 281 (92%) provided successful Pyrosequencing results (see figures  High PCR success rate with bisulfite converted DNA and  High Pyrosequencing success rate of predesigned PyroMark CpG Assays). The high-performing assays eliminate the need for tedious assay optimization and shorten the time to reliable results.
See figures


Assays for analyzing gene-specific CpG sites
PyroMark CpG Assays enable methylation analysis of specific targets across the genome. Our database now contains over 84,000 predesigned assays covering more than 80% of gene-specific CpG islands for the human, mouse, and rat genomes. These assays are designed using a carefully tailored algorithm that generates optimal assay design based on bisulfite converted DNA. As such, these fast and easy-to-order assays minimize time spent on assay optimization and maximize analysis success. PyroMark CpG Assays expand the streamlined Pyrosequencing workflow to enable methylation analysis of virtually any CpG island in the human, mouse, or rat genome (see figure  Analysis of ESR1).

Assays for methylation array validation
Hybridization arrays enable high-throughput discovery of variations in DNA methylation, but the results must be experimentally verified to confirm that the correct variations are identified. Pyrosequencing is a cost-effective technique highly suited for this purpose, providing quantitative analysis of short- to medium-length sequences with high accuracy. We now offer predesigned PyroMark CpG Assays for validating results from HumanMethylation450K and MethylationEPIC BeadChip experiments. These new predesigned assays for methylation array validation offer a convenient way to verify experimental results, and determine exact methylation levels of CpG sites of interest, without the need to manually design a separate assay for each individual target of interest. Our dedicated search page lets users simply enter the CpG loci identification numbers (CG#) of interest to easily search our assay database and order ready-to-use PyroMark CpG Assays.

The advantages of GeneGlobe
QIAGEN's GeneGlobe Web portal is a one-stop online interface that grants access to our extensive range of assay technologies in a gene- or pathway-specific context. GeneGlobe brings together state-of-the-art search and design algorithms, up-to-date sequence data, and our innovative technologies to facilitate discovery. Simply enter a search term for the gene or pathway of interest, and let GeneGlobe show you how to advance your research in epigenetics, or any other area of interest (see figure  Ready-to-use methylation assays).
See figures


PyroMark CpG Assays include PCR and sequencing primers for quantification of DNA methylation by Pyrosequencing. A biotinylated PCR product is generated from the bisulfite converted DNA template and is bound to streptavidin-coated Sepharose beads. The beads are captured with the vacuum tool on the PyroMark Vacuum Workstation, where they are thoroughly washed and subsequently denatured, generating single-stranded DNA suitable for Pyrosequencing. This template DNA is released into the Pyrosequencing reaction plate containing the sequencing primer provided with PyroMark CpG Assays. After primer annealing, the plate is placed into the PyroMark instrument, and the sequencing reaction is started.


PyroMark CpG Assays enable highly accurate, quantitative methylation analysis of gene-specific human, mouse, and rat CpG sites as well as targets from the HumanMethylation450K and the MethylationEPIC BeadChip for epigenetics research.

Supporting data and figures


In which format can PyroMark CpG Assays be ordered?
Pyromark CpG Assay can be ordered in tubes or on 96 well plates. PyroMark CpG Assays 96 well require a minimum order of 24 assays per plate.
FAQ ID -2824
Where can I order the Streptavidin Sepharose beads for pyrosequencing?
The recommended Streptavidin Sepharose High Performance beads for pyrosequencing can be ordered at GE healthcare with the catalog no 17-5113-01.

The PyroMark Q48 Autoprep protocol uses magnetic streptavidin-coated Sepharose® beads (PyroMark Q48 Magnetic Beads), which bind to the biotinylated PCR strand.

PyroMark Q48 Magnetic Beads can be ordered at QIAGEN with the catalog no 974203.

FAQ ID -2850
Can I order the nucleotides from PyroMark Gold Reagents separately?
The nucleotides can only be ordered as part of the PyroMark Gold Reagents which also contain enzyme and substrate mix.
FAQ ID -2827
How many nucleotides of a homopolymer can be resolved in pyrosequencing?
In the range of 3-5 bases can be resolved depending on the sequence context and base. If it is possible sequencing of a homopolymer of more than 3-5 nucleotides should be avoided by resetting the sequencing primer.
FAQ ID -2871
What is the reason for a high substrate peak in the pyrosequencing pyrogram?
Usually pyrophosphate or dATP/ATP contamination in the sample or in the buffer can cause a high substrate peak. Large amounts of pyrophosphate are generated in the PCR reaction and might be carried over to the sequencing reaction. Check the PyroMark buffers and reagents and use new ones.
FAQ ID -2879
What are the features of PyroMark CpG Assays e.g. in terms of design, validation?
PyroMark CpG Assays are genome-wide, pre-designed methylation assays for pyrosequencing analysis. An optimized design algorithm was used for highly specific assay design and advanced CpG methalytion results.
FAQ ID -2821
Which kits can be used in combination with the PyroMark CpG Assays and PyroMark Custom Assays?
QIAamp/DNeasy Kits can be used for DNA isolation, EpiTect Bisulfite Kits for DNA conversion, PyroMark PCR Kit for PCR amplification, EpiTect Control DNA Set for PCR controls, and PyroMark Gold Q24 Reagents or PyroMark Gold Q96 Reagents for the sequencing reaction.

Depending on the platform used, the following reagent kits are required for Pyrosequencing:

PyroMark Q96 ID and MD: PyroMark Gold Q96 Reagents

PyroMark Q24: PyroMark Gold Q24 Reagents

PyroMark Q24 Advanced: PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents

PyroMark Q48 Autoprep: PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents

FAQ ID -2822
How do I reduce background peaks in the pyrosequencing pyrogram?
There are several reasons for a high assay background; the template can form secondary structures which are extended or the primers itself form dimmers which serve as template. Perform accurate sequencing controls (e.g. PCR or sequencing primer only) as recommended in the PyroMark User Manual to observe this kind of background. In addition, an unspecific priming of primer to template or unspecific annealing of sequencing primer to template might also be a background cause. Please check your complete primer design and if needed, perform a redesign. Try to lower the primer concentration as possible to avoid excess of primer.
FAQ ID -2877
How are the PyroMark CpG Assays shipped and stored?
Pyromark CpG Assays are shipped lyophilized at ambient temperatures (20-25°C) and should be stored at -20°C either reconstituted or lyophilized. Repeated freeze-thaw cycles should be avoided. When stored under these conditions, the reconstituted product can be kept for at least 18 month from the date of receipt without reduction in performance.
FAQ ID -2816
How are the PyroMark CpG Assays reconstituted?
The PyroMark CpG Assay is reconstituted as a 10x PCR Primer Set in 550µl TE, pH 8.0 and the 10x Sequencing Primer is reconstituted in 1175µl Annealing Buffer if using the PyroMark Q24, 880µl if using the PyroMark Q96 ID, and 1175µl if using the PyroMark Q96 MD.
FAQ ID -2817
How many times can vacuum troughs be re-used with the PyroMark Vacuum Preparation Stations?
There is no precise recommendation how many times these troughs on the PyroMark Vacuum Preparation Stations (Q24 and Q96) can be re-used. It depends on the individual handling and cleaning (with water).
FAQ ID -2848
What concentration should be used for the sequencing primer in pyrosequencing?

Usually the sequencing primer is used at 0.3µM in annealing buffer but some assays might require additional optimization of the sequencing primer concentration.


For PyroMark Q24 and PyroMark Q96 MD the final concentration of the sequencing primer is 0.3µM and for PyroMark Q96 ID 0.4µM.

The PyroMark Q48 Autoprep dispenses the sequencing primers for annealing. The final concentration of sequencing primers in a well is 0.8µM, but may be adapted to optimize assays.


FAQ ID -2826
Does QIAGEN offer a design of Custom PyroMark CpG Assays?
No, QIAGEN does not design any Custom PyroMark CpG Assay. Customers have the possibility to order pre-designed, genome-wide PyroMark CpG Assays or order a user-designed assay (e.g. with the PyroMark Assay Design Software or assays known from previous projects or from the literature).
FAQ ID -2818
Will the primer sequence for the PyroMark CpG Assay be provided?
Primer sequences for PyroMark CpG Assays are not provided, they are proprietary.
FAQ ID -2823
Which purity grade is recommended for pyrosequencing primers?
Only the biotinylated primer needs to be HPLC purified whereas the other primers require standard desalting only.  Pyrosequencing primers can be ordered here.
FAQ ID -2832
What is the reason for split peaks appearing in between dispensations on my pyrosequencing pyrogram?
The PyroMark cartridge needle can be blocked or damaged. Clean the cartridge or exchange with a new one. Check for correct reagent cartridge and cartridge method used in the run. Check if the reagent cartridge cover was closed properly. Make sure that the cartridge was dry after cleaning because nucleotide droplets might be caught at the needle tip and fall down at any time. or exchanged.
FAQ ID -2881
Is the CpG software included in the PyroMark instruments to study methylation status?
The PyroMark Q24 software and the new PyroMark Q96 ID software version 2.5 support CpG analysis in the CpG mode. The ORACLE-based PyroMark Q96 ID software version and PyroMark Q96 MD software do not support CpG analysis. In this case an independent, additional software is needed which is the PyroMark CpG software version 1.0.
FAQ ID -2842
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
What is the reason for signals ceasing in the middle of a pyrosequencing run?
The cartridge needle can be blocked or damaged causing a dispensation error. Clean the cartridge following the guidelines or repeat the run with a new cartridge. On the other hand if high amounts of template have been used resulting in very high signals (>100 RLU), the substrate for the sequencing reaction might be depleted. In this case template conditions should be optimized.
FAQ ID -2875
What is the recommended amplicon size for CpG assays?
The amplicon length should be short (<200bp). This is critical especially for DNA from FFPE tissue which is often degraded by the fixation so that short fragments are easier to amplify. Moreover, the DNA suffers from harsh bisulfite treatment and might receive further double strand breaks. Therefore the amplicon size should be kept as short as possible.
FAQ ID -2825
Can unused wells in a pyrosequencing plate be used in the next run?
In principle it’s possible to use so far unused pyrosequencing wells for the next run and leave the already used wells empty. However, due to contamination risk when cleaning and handling plates QIAGEN does not recommend this.
FAQ ID -2872
How do I set up a PyroMark CpG Assay?
All relevant information regarding PyroMark CpG Assay setup can be found on the Gene Globe website. The Sequence to Analyse and dispensation order should not be copied manually to create a new assay. Instead, the assay file should be downloaded from the web and opened in the PyroMark CpG software, PyroMark Q96  ID v2.5 (or higher) software, and PyroMark Q24 Software in order to keep important software settings.
FAQ ID -2814
How do I prevent a drifting baseline in my pyrosequencing pyrogram?
Let the PyroMark instrument warm up (about 60 minutes) to adapt to room temperature before use. Make sure the ambient room temperature is within range 18-28°C.
FAQ ID -2878