Course Identification

Real-time PCR has become a robust and a routine approach for detecting and quantifying nucleic acids. Thus, real-time PCR is used for measuring the expression of genes, validating the results from micro-array experiments, genotyping, analyzing DNA variations (such as DNA SNPs) as well as for monitoring various bio markers in microbiology and oncology.

In this lab course we will discuss the fundamentals of real-time PCR and will review many of its applications. The participants will have an opportunity to practice quantification of gene expression. We will conduct experiments with samples containing  low copy number of the target DNA by preforming pre-amplification to overcome such a common limitation. Towards this end, we will prepare RNA for mRNA expression analysis and RNA for micro-RNA (miR) expression analysis. Subsequently, two classes of chemistries will be introduced. The first utilizes the dsDNA binding fluorescent probe SYBR and the other technology utilizes a target-specific probe that fluoresces upon hybridization and hydrolysis (TaqMan). Most importantly, we will practice two methods for data analysis (the standard curve-assited mode of analysis and the DDC method). We will demonstrate primer design, and we will discuss and practice strategies for data analysis and statistical evaluation of the data. Finally, we will discuss various aspects of experimental design and requirements of adequate report of results according to the recently established MIQE guidelines.

Upon successful completion of this course students should be able to:

1. Discuss the use of real-time PCR as a reliable and sensitive method for quantifying nucleic acids and its applications. The students will be able to appreciate the approaches to detect low-copy number of a target sequence of choice.
2. Discuss algorithms for primer design.
3. Experiment (hands on) to get relative quantification of selected cellular mRNAs and miRs.
4. Collect data as well as differentiate between the various methods of analysis.
5. Discuss the need for suitable standards to guide proper practice and data communication. In this regard, appreciate the need to adhere to the well-defined MIQE (Minimum standard for publication of qPCR Experiments) criteria.
6. Obtain and appreciate tools for statistical analysis.
7. Translate the acquired the knowledge. Thus, allow to adapt, in one?s own systems of choice, proper technical and analytical methodologies that are reliably used in the practice of real-time PCR.