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.
Owing to the unfortunate COVID-19 pandemic, we have revised a well-established laboratory course by focusing on the theory of qPCR that will be followed by a short experimental module. Thus, lectures will be given online through Zoom sessions and practical sessions will be carried out in the students’ laboratories and terminated at the FGS laboratory. Students will analyze their own experimental data. In this revised course, 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). Importantly, we will learn about the importance of primer design, and we will practice extensively (online) 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.