qPCR-based gene expression analysis (for large scale projects)
Reverse transcription quantitative PCR (RT-qPCR) distinguishes itself from other methods available for gene expression in terms of accuracy, sensitivity, and fast results. Because of this, the technology has established itself as the gold standard for medium throughput gene expression analysis. Biogazelle's founders have more than 10 years of experience in real-time PCR experiment design, assay development and data analysis. They wrote one of the most influential papers on normalization of gene expression and on data analysis (together cited more than 2000 times). Biogazelle is your preferred partner for medium or large scale gene expression analysis using the SYBR Green detection chemistry!
Background
Primer design and validation
Custom primer design is performed using the RTprimerDB that embraces an integrated primer in silico evaluation system to detect potential assay features that could negatively influence the assay's efficiency or specificity. 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) at the primer annealing sites. If the RTprimerDB cannot be used, 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. Extensive empirical validation of the primer pairs follows thorough in silico quality control. 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 to minimize 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
- RNA 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.