Quantitative Real-time Polymerase Chain Reaction (qPCR) Methods for Abundance and Activity Measures
Vibeke Børsholt Rudkjøbing, Tine Yding Wolff and Torben Lund Skovhus
from: Applications of Molecular Microbiological Methods (Edited by: Torben L. Skovhus, Sean M. Caffrey and Casey R.J. Hubert). Caister Academic Press, U.K. (2014)
Quantitative real-time polymerase chain reaction (qPCR) is a method by which DNA and RNA target molecules can be analyzed in real-time and quantified. The method has a wide range of applications, and its use has become increasingly popular in different scientific and commercial fields. This method is based on amplification of target molecules in the presence of fluorescent dyes that enable detection. Despite this relatively simple principle there are many options for customizing and optimizing the reaction, both in terms of overall experimental setup and choice of dye and DNA/RNA target. Besides an introduction to the fundamentals of qPCR, this chapter contains a description of theory and strategy and demonstrates how to use qPCR to determine microbial abundance and activity. Abundance measurements are generally performed by targeting DNA and typically include use of standard dilution series in order to obtain quantitative measurements. Conversely, the activity of microorganisms is determined by gene expression profiling, and the target is typically cDNA synthesized from RNA molecules. Such experiments generally do not provide absolute measurements, but instead are performed as a relative quantification, where the measurements of the target gene are related to a reference gene. The qPCR method has many advantages; chiefly it is a culture-independent method that offers great simplicity and flexibility along with a rapid turnaround time of a few hours. Additionally, the method has relatively low instrumentation demands. However, there are some critical limitations and considerations to the method, which are described in the chapter read more ...