Real-time PCR (qPCR) as a robust and a routine approach for detecting and quantifying nucleic acids is used for measuring gene expression, validating the results from high-throughput experiments, genotyping, analyzing DNA variations (such as DNA SNPs) as well as for monitoring various bio markers in microbiology and oncology. Practicing qPCR and publishing the results of such experiments requires one to meet the MIQE guidelines (Bustin, SA et al., 2009).
MIQE guidelines, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines, is a set of guidelines that describe the optimal experimental practice for obtaining reliable results and the minimum information necessary for evaluating qPCR experiments. In addition, MIQE guidelines help ensure the integrity of the scientific literature. Thus, included is a checklist to accompany the initial submission of a manuscript to the publisher aimed at promoting consistency between laboratories, and increased experimental transparency.
In this lab course we will bring into consideration the MIQE guidelines to discuss the fundamentals of real-time PCR and we will review many of its applications. Subsequently, two classes of technologies will be introduced. The first utilizes the dsDNA binding fluorescent probe SYBR and the other technology utilizes a target-specific probes (“TaqMan”). Two modes of data analysis will be evaluated and compared. The participants will have an opportunity to practice quantification of gene expression in extreme cases that are characterized by low-copy number of a target. In particular, to cope with such challenges, we will prepare cDNA and perform pre-amplification followed by qPCR and data analysis. In addition, we will learn how to analyze data of microRNA (miR)-target interactions obtained in pull-down assays. Importantly, we will practice primer design, and over two full sessions we will discuss and practice strategies for statistical evaluation of qPCR data.