qPCR-based gDNA copy number screening (for large scale projects)
Copy number changes under the form of deletions and duplications are known to be involved in numerous (human) genetic disorders. Hence, it is not surprising that a wide spectrum of laboratory methods has been developed to identify these copy number changes. qPCR based copy number screening using SYBR Green has been less frequently used in comparison to other techniques such as arrayCGH, but recent high-throughput developments hold the promise to take this application to the next level. qPCR has many advantages over alternative methods, such as its high sensitivity, its accuracy, its flexibility, its relatively low consumable and instrumentation costs, its fast turnaround and assay development. Biogazelle's experienced service unit has access to state-of-the-art technologies for large-scale qPCR based copy number screening, making it your preferred partner!
Background
Primer design and validation
Custom primer design is performed using several software tools for the design and in silico evaluation of high-quality assays based upon in-house validated parameters (D'haene et al., Methods, 2010). Potential assay features that could negatively influence the assay's efficiency or specificity are avoided. The in silico validation is necessary (1) to guarantee that the primers are specific, (2) to avoid secondary structures, and (3) to avoid single nucleotide polymorphisms (SNPs) and copy number polymorphisms at the primer annealing sites. After thorough in silico quality control follows an extensive empirical validation of the primer pairs. First, amplification efficiencies are calculated based upon the generation of standard curves using 6-point dilution series. Data analysis is done in Biogazelle's qbasePLUS software. Subsequently, melt curve analysis and microchip electrophoresis are used to verify the specificity of the PCR reactions.
Experimental design & plate lay-out
The experimental design is optimized for each project to pursue the highest standards of qPCR-based research. Biogazelle is extremely flexible to accommodate your needs and works out the optimal design based on years of experience. The sample maximization method (Hellemans et al., Genome Biology, 2007) is preferentially used because it minimizes technical plate-to-plate variation. The entire qPCR workflow is performed using rigorous MIQE compliant procedures, high-throughput laboratory facilities, and state-of-the-art technologies. These high-throughput laboratory facilities enable large scale experiments with exceptional value for money. Data analysis is performed using Biogazelle's qbasePLUS software, which is built upon a state of the art and proven quantification model (Hellemans et al., Genome Biology, 2007) including PCR efficiency correction, multiple reference gene normalization, inter-run calibration and error propagation, and offers numerous tools for quality control. Further statistical analysis can be offered upon request.
Different steps/processes made by Biogazelle
Primer design and validation
- In silico primer design and validation
- Empirical primer validation using serial dilutions and microchip electrophoresis
Full experiment
- Sample reception + quality control with UV-Vis spectrophotometer and microchip electrophoresis
- Sending of an acknowledgment of receipt
- DNase I treatment and cDNA synthesis
- Reaction set up
- Measurements in real-time PCR instrument
- Data analysis in Biogazelle's qbasePLUS
- Sending of a full report
List of Cq values
Result table (normalized relative quantities with error values), ready for downstream bio-statistical analysis
qbasePLUS real-time PCR data analysis software experiment file (for client reanalysis)
RDML file for publication or data storage
Pricing
We are extremely flexible to accommodate your needs and we guarantee exceptional value for money. Please contact our service unit (service@biogazelle.com) for more information or a quotation. To provide pricing it is convenient to have an idea about the number of samples you would like to analyze.