Review ArticleAccurate and objective copy number profiling using real-time quantitative PCR
Introduction
Copy number changes under the form of deletions and duplications are known to be involved in numerous human genetic disorders. Moreover, each individual’s genome embodies several copy number polymorphisms of various sizes which are thought to contribute to normal phenotypic variation and susceptibility to multifactorial disease [1], [2]. Hence, it is not surprising that a wide spectrum of laboratory methods has been developed to identify these copy number changes. Well known and widely applied techniques include conventional karyotyping, fluorescent in situ hybridization (FISH) analysis, microarray-based copy number screening, multiplex ligation-dependent probe amplification (MLPA), and quantitative PCR (qPCR) [3], [4]. Each method is characterized by particular (dis)advantages and the choice for a given technique largely depends on the application, required resolution, flexibility, workload, and cost. Conventional karyotyping allows the detection of structural variations across the entire genome, but it is limited in resolution (>5–10 Mb). FISH analysis for targeted regions has been used in a routine setting for many years, and requires either metaphase chromosomes (similar to karyotyping) or interphase nuclei (resolution approximately 100 kb). Microarray-based copy number profiling has improved the resolution in the last decade and facilitated the detection of much smaller copy number changes [4]. The most recent high density targeted arrays even achieve a resolution of a few base pairs. For patients with mental retardation or other complex phenotypes, genome wide copy number profiling using microarrays proves to be the most suitable approach to reveal the underlying molecular defect [5]. In contrast to the research driven race for ever increasing resolution, the majority of diagnostic tests for genetic disorders are restricted to the targeted screening of the relevant disease genes and their surrounding regulatory landscapes. In the latter context, focused copy number screening methods are preferentially used, such as MLPA, targeted microarrays, and qPCR.
With real-time qPCR, the PCR product accumulation is measured in real-time resulting in a sigmoidal amplification curve. Several detection chemistries are available to measure product accumulation, including hydrolysis probes, molecular beacons, dual hybridization probes and double stranded DNA specific binding dyes. There is a relationship between the moment that the fluorescent PCR signal increases above the background and the initial amount of input DNA; larger amounts of input material will result in lower quantification cycle (Cq) values. The Cq value represents the fractional PCR cycle that is characteristic for the amplification curve (e.g. where increase in fluorescence is maximum) or at which the fluorescence crosses a certain threshold. qPCR has many advantages over alternative methods, such as its low consumable and instrumentation costs, fast turnaround and assay development time, high sensitivity and open format (independent of a single supplier). To date, qPCR is the golden standard for gene expression analysis. For copy number determination, qPCR has been less frequently used, but recent developments hold the promise of taking this application to the next level.
In this chapter we provide all relevant information required to use qPCR for copy number analysis.
Section snippets
Selection of regions
The overall accuracy of an answer to a specific research or diagnostic question largely depends on the number of qPCR assays, their position relative to the disease locus, and the spatial interval between subsequent amplicons. First, the genes or intergenic regions of interest are selected. The number of selected genes usually affects the number of assays included per gene. When a large series of genes needs to be screened, the number of assays per gene is often restricted to one or two due to
Concluding remarks
Real-time quantitative PCR is a perfectly suited method for the detection of copy number variations in targeted regions because of its low screening cost and fast turnaround time. Adhering to the following general guidelines increases the quality and reliability of the determined copy numbers. First of all, take care of the preparations: assay design and validation, as well as experiment design. Secondly, perform as much quality controls as possible along the entire workflow: QC on the assay
Acknowledgments
This study was supported by Specialisatiebeurs from Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) (B.D.); 1.2.843.07.N.1 from the Research Foundation Flanders (FWO) (J.H.); 01209407 from BOF-UGent (J. VDS.).
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2024, Molecular Aspects of MedicineThe relationship between MUC19 copy number variation and growth traits of Chinese cattle
2023, GeneCitation Excerpt :In this experiment, real-time quantitative PCR (qPCR) technique was used to detect copy number variation in the MUC19 gene. The principle of the technique is to quantify the gene to be tested relative to the internal reference gene, and then use the detection values of both to infer the copy number status of the gene to be tested (D'haene et al., 2010). The amplification system for qPCR is 10 μL:1μL template DNA;0.2 μL forward primers and reverse primers, 5 μL SYBR ®PreMix Ex Taq TM II and adding ddH20 to 10 μL.