Course Identification

Real-time PCR (qPCR) has become a robust and routine method for detecting and quantifying nucleic acids. The main uses of qPCR are in measuring the expression of genes, validating the results from microarray and RNA-seq experiments, genotyping, analyzing DNA variations (such as DNA SNPs) as well as for monitoring various biomarkers in microbiology and oncology.

 We will discuss the fundamentals of real-time PCR and review many of its applications. Participants will have an opportunity to learn about the quantification of gene expression, among other relevant topics. In particular, we will discuss the preparation of RNA for mRNA expression analysis and for micro-RNA (miR) expression analysis. Then, two different chemistries will be introduced. The first utilizes the dsDNA binding fluorescent probe SYBR and the other utilizes a target-specific probe that fluoresces upon hybridization and hydrolysis (TaqMan being one commercially available product).  On the practical part, we we introduce methods for quantifying low copy number (rare) mRNA targets by pre-amplification which should preceed the real-time PCR stage. Studentds will perform these protocols and runs their own plates. Importantly, we will learn about the importance of primer design, and we will practice extensively strategies for data analysis (the standard curve-based approach versus other approaches). In addition, we will discuss various aspects of adequate experimental design and the requirements for reporting results according to the recently established MIQE guidelines, which ensure proper practice and the reproducibility of scientific results. This will be followed by an introduction to statistical analysis of the data. Finally, given the use of qPCR in microbiology in order to detect various pathogens, we will explain how to detect the SARS-CoV-2 by using a real-time PCR-based assay that requires specially designed hydrolysis probes.

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.

2. Discuss algorithms for primer design.
3. Learn about relative quantification of selected cellular mRNAs and miRs.
4. 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 real-time PCR.