Day 3 – Wednesday 17th November 2010

Post Conference Workshop - Experimental Design and Quality Control in qPCR Experiments

Course Tutor: Mikael Kubista, TATAA Biocenter & Institute of Biotechnology at the Czech Academy of Sciences

Mikael was among the pioneers who developed real-time PCR. Starting in 1991 his laboratory developed dyes and probes for real-time PCR and founded LightUp Technologies as Europe’s first company focusing on real-time PCR based human infectious disease testing. His team then developed experimental approaches for accurate measurements of expression levels, and they pioneered the fields of single cell expression profiling and multidimensional expression profiling by real-time PCR. Mikael also developed methods and approaches to analyze gene expression data and co-founded MultiD Analyses that develops the popular software GenEx for processing of real-time PCR data. Working as advisor for Unesco he introduced real-time PCR in Africa and in the Middle East. In 2001 Mikael founded the TATAA Biocenters as global leading service providers and organizers of hands-on training in real-time PCR. Regular training courses are held all over Europe, Africa, Asia and in the US. The TATAA courses world-wide are supported by leading instrument manufacturers and reagents suppliers in the real-time PCR field. Currently Michael’s work focuses on circulating tumor cells and the development of guide lines, where he recently co-authored the MIQE guidelines, and quality control for qPCR analysis and the pre-analytical steps, which is done within SPIDIA. Mikael is also advisor to several biotech and pharmaceutical companies.

1 Day Experimental Design and Quality Control in qPCR Experiments 

9am – 5pm including Lunch and refreshments

 The key to successful qPCR analysis is arguably quality experimental design that balances statistical significance and experimental cost. Performing a fully nested pilot study GenEx estimates the variance contributions from the different experimental steps, advising you which steps may be improved to enhance data quality and were technical replicates shall be performed. It also indicates the number of subjects needed to achieve a desired resolution. Too few subjects and you may not be able to prove or disprove your hypothesis, while too many subjects may improve resolution beyond what is practically relevant and money is wasted. Further requirement for success is high performance of the equipment being used. Mal-performing dispensers or qPCR instruments may compromise a study. This can be avoided by using proper standard operating procedures including quality controls. Finally, data shall be reported according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines such that results can be properly evaluated and easily reproduced.

• Absolute quantification, qPCR standard curve, Reverse calibration, Limit of detection
• Experimental design, Noise contributions to RT-qPCR analysis (nested ANOVA), cost-performance optimization of experiments 
• Sample size estimations (Power testing)
• Selecting reference genes (geNorm, Normfinder)
• qPCR data pre-processing, Outlier detection. Relative quantification, Comparison of groups (parametric and non-parametric methods)
• Expression profiling, missing data treatment, scaling of data, Un-supervised clustering of genes and samples (hierarchical clustering, self-organized maps, Principal Component Analysis), Supervised clustering of samples (Artificial neural network)
• Standard operating procedures and quality control
• The MIQE guidelines
• Exercises