Pyro Q-CpG™ is an accurate and fast analysis method for quantifying CpG methylation in epigenetic studies. Since its inception in 2002, it has established itself among leading researchers as the gold standard of DNA methylation analysis. Pyro Q-CpG has revealed correlation between DNA methylation of genes to tumor type and gene expression, measured the response to treatment with demethylating agents, as well as changes in methylation state in relation to tumorigenesis and tumor progression, genetic imprinting and exposure to environmental toxins.
The most appreciated property of Pyro Q-CpG is its highly reproducible quantification of methylation frequencies in individual consecutive CpG sites, enabling reproducible measurement of even small changes in methylation levels. The reproducibility of Pyro Q-CpG is a result of a quantitative measurement principle, inherent quality controls (QC), and few processing steps. QC is inherent because CpG sites are presented in the context of the DNA sequence, and controls for completion of the bisulfite conversion step can be integrated into the analysis process.
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Quantitative DNA methylation analysis by Pyrosequencing enabled us to distinguish tumor-specific methylation from “noise” for p16, MGMT and CYGB promoters. This contrasts with previous published work using MSP, and demonstrates the significance of a quantitative rather than qualitative methylation assay.
Dr. Triantafillos Liloglou, University of Liverpool Cancer Research Centre |
Based on Pyrosequencing® technology, Pyro Q-CpG analyzes and presents the individual methylation levels of multiple consecutive CpG sites in the context of the DNA sequence. Single CpG sites can be analyzed as easily as multiple sites, and methods are established for estimating global methylation.
A real-world example of methylation analysis by Pyro Q-CpG
The following example illustrates methylation analysis of the MLH1 gene. Nineteen consecutive CpG sites in the promoter region were analyzed on samples isolated from head and neck tumors. Pyrosequencing analysis performed on this 130 bp sequence takes 130 minutes for up to 96 samples. Thereafter, analysis and quality control of methylation takes 1 minute for up to 96 samples.
Using Pyro Q-CpG, regions covering multiple CpG sites can be analyzed in a single reaction. This analysis provides individual, reproducible quantification for all 19 CpG sites. In the Pyrogram® trace above, two regions, indicated in bold text in the sequence, are magnified to illustrate the individual methylation results. Methyltion levels in yellow indicate CpG sites that have not fully passed quality control.
Data on MLH1 kindly provided from Dr. Triantafillos Liloglou, University of Liverpool Cancer Research Centre,
Aintree Hospital Maxillofacial Surgery Dept.
The Pyro Q-CpG Software makes DNA methylation analysis available for all Pyrosequencing platforms.
Pyro Q-CpG from QIAGEN offers these advantages for methylation analysis:
- Reproducible quantification of individual, consecutive CpG sites enables discrimination of
even small changes in methylation levels
- Versatile for a range of analyses, from single CpGs to global methylation
- Easy to communicate data: methylation levels are presented in sequence context
- Quality control: sequence context, comparison to expected methylation levels, and built-in QC for bisulfite treatment
- Suitable for analysis of fresh-frozen, fixed and paraffin-embedded specimens
- Robust and flexible assays, ready-made and custom-designed
- Fast turnaround: 96 samples analyzed in parallel in 1 hour after PCR
Pyro Q-CpG is a complete system for methylation analysis comprising instrumentation, chemistry, software, assays and analysis services. Based on Pyrosequencing® technology, Pyro Q-CpG builds on the solid scientific and technical knowledge of our own staff, as well as routine collaborations with cancer geneticists.
Instrumentation
Pyro Q-CpG can be performed on any Pyrosequencing instrument.
Pyro Q-CpG Software
Pyro Q-CpG Software has been developed to address applications that benefit from high-quality and high-throughput analysis of DNA methylation. This is achieved by quantitative sequence-based analysis of multiple, consecutive CpG sites in 96 samples in parallel, enabling an individual to analyze and process many thousands of samples and CpG sites to provide statistically reliable data.
Functions that are essential for high throughput and data reliability have been incorporated into Pyro Q-CpG Software. After the Pyrosequencing run, analysis of ninety-six samples takes less than one minute, even when each sample contains 20 or more CpG sites. Another key improvement is the automatic quality control of each sample for completion of bisulfite treatment, eliminating the need for manual inspection and estimation of levels of non-converted DNA.
With the overview feature, the user can easily navigate through methylation results, mean values, methylation patterns and quality scores for each sample as well as individual CpG sites.
Pyro Q-CpG software is compatible with all existing Pyrosequencing instrument platforms. It can be installed and run in parallel with the current instrument control software.
Take a 3 minute tour to learn more about the Pyro Q-CpG Software
How Pyro Q-CpG works, step-by-step
Pyro Q-CpG quantifies methylation in explicit sequence context, and is both fast and easy to perform. Assay design is flexible, since the distance from the first base to be sequenced can be varied, and therefore the primer can usually be positioned in a region free of CpG sites. In addition, there are four options for design; the assay can be performed in forward or reverse orientations on either the top or the bottom strands.
| Step |
Time |
| Typical workflow for analysis of CpG methylation for 96 samples in parallel |
| Bisulfite treatment |
3 hours hands-on, overnight incubation |
| PCR amplification |
2 hours |
| Preparation of single-stranded DNA |
5 minutes hands-on, 15 minutes total |
| Pyrosequencing |
1 minute/dispensation, typically 30 min |
| Calculation of methylation at each CpG site |
1 minute |
Typical workflow for analysis of CpG methylation for 96 samples in parallel
The approach to analyze methylation exploits one of the most powerful attributes of Pyrosequencing, namely its readout (the peaks in the Pyrogram). Unlike dideoxy (Sanger) sequencing, the peak heights produced by Pyrosequencing quantitatively report the proportions of alleles.
Unmethylated C (red) and methylated C (green) are differentiated by bisulfite treatment and PCR. The ratio C:mC at each CpG site (peaks in yellow column) is measured in sequence context.
C not followed by G acts as control for the bisulfite step (blue column).
Sequence context is an important control since bisulfite-treated, PCR-amplified DNA is AT-rich, which decreases sequence variation. Pyro Q-CpG therefore guarantees that the correct sequence was analysed.
Pyro Q-CpG assays can contain an internal control for bisulfite treatment. C that is not followed by G in sequence is not methylated, and should therefore be fully converted to T by bisulfite and PCR. To confirm this, all templates should show only T and zero C in this position (blue column).
Pyro Q-CpG is practical in terms of starting material and throughput. DNA is readily analysed from both fresh frozen tissue as well as the short PCR fragment sizes that are typical of paraffin-embedded tissue, where restriction fragment analysis would be difficult. The analysis takes about 1 hour for 96 specimens in parallel, at a fraction of the cost and time of the equivalent dideoxy (Sanger) sequencing reactions.