Development of a Real-Time Quantitative PCR Assay: Choices and Strategies
Tim Hunter, BS, Manager, VCC DNA Analysis Facility; Manager, UVM Microarray Facility, University of Vermont
Quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used, rapid and sensitive method for the quantification of mRNA. It has become the "gold standard" for both pathogen detection and gene expression studies and is the method of choice for corroborating microarray data. This course will walk through the steps in the process of developing an assay including experimental design, probe/primer guidelines, RNA extraction, quantitation, assessment of RNA integrity, gDNA elimination, cDNA synthesis, qPCR, and quantification strategies. Emphasis will be placed on incorporating quality control measures at each step and the potential to develop multiplexed assays. Examples of the validation of microarray data employing qRT-PCR through different chemistry and quantification strategies will be presented and discussed.

Who should attend: This course will be valuable for anyone interested in using this technology for gene expression, microarray validation, pathogen detection, or genotyping, and will be targeted for all levels of experience.

Why attend: To gain an understanding of the necessary requirements in the development of gene expression assays and where variation can be introduced.

Short Course Two

Statistical Analysis of Real-Time PCR Data
Joshua S. Yuan, M.S., Ph.D. Candidate, Genomics Scientist, The UTIA Genomics Hub, The University of Tennessee, Knoxville
C. Neal Stewart Jr., Ph.D., Racheff Chair of Excellence in Plant Molecular Genetics, Department of Plant Sciences, The University of Tennessee, Knoxville
Real-time PCR has been broadly applied in biomedical sciences, especially for high-throughput gene expression studies and functional genomics data confirmation. However, the accuracy and reproducibility of the technology has become a major concern of the scientific community. Proper data analysis is the key to the precise quantification and accurate interpretation of the real-time PCR data. In many publications for transcriptome studies, the data processing procedures for the analysis of quantitative real-time PCR are still lacking; specifically in the realm of appropriate statistical treatment. Confidence interval and statistical significance considerations are not explicit in many of the current data analysis approaches. We therefore developed this tutorial based on our BMC bioinformatics article (Yuan, et al., BMC Bioinformatics 7:85) and several other recent publications. The tutorial’s purpose is to enable end-users to:

• better understand the mathematical attributes of the real-time PCR data,

• be aware of the recent advancements in statistical analysis of real-time PCR data,

• be able to obtain high-quality results with proper statistical estimations,

• be familiar with the practical programs and software based on statistical models

• perform accurate data analysis when low amplification efficiency occurs,

• recognize the standards for real-time PCR data analysis procedures.

Who should attend: Researchers, core directors, clinicians, post-docs and students interested in gene expression studies or real-time PCR will learn about important aspects and recent developments of real-time PCR statistical analysis. The tutorial is designed for the biologists as well as the bioinformaticians and statisticians interested in processing molecular biology data. The tutorial will briefly cover necessary genetics, molecular biology and statistics backgrounds. An introductory knowledge of statistics will be helpful, yet is not required. Most of the programs are very simple and are designed for biologist users. A laptop computer with SAS installation will be helpful, but not required.

Why attend: Scientists and editors alike are realizing that inappropriate data processing and statistical analysis of genomics in general and real-time PCR data specifically can lead to faulty conclusions. End-users eagerly desire biologically correct interpretations and their papers published! Statistical analysis of real-time PCR data has been declared ‘a new challenge in gene quantification analysis’. The tutorial provides a practical and comprehensive introduction to common statistical models and programs used for real-time PCR data analysis. The tutorial will also help researchers to be familiar with the standards required for real-time PCR data procedures.