Quantitative polymerase chain reaction using the comparative C q method.

Quantitative PCR (qPCR) is considered the gold standard for molecular DNA quantification and can be used for a wide range of techniques from comparing gene expression levels to quantifying DNA copy number variation. The strengths of this assay include sensitivity to a wide range of expression levels, low starting template requirement, which is important when samples are scarce, and quick turnaround time. However, there are many variables to consider when performing qPCR: including (1) starting materials (e.g., tissues, cells, or genomic DNA), (2) fluorescent detection (e.g., SYBR green dye, fluorescent probes, or multiplexed assays), and (3) analysis methods (e.g., simple equations with single reference genes or complex algorithms with multiple reference genes). This chapter will introduce the process of designing an experiment while avoiding common mistakes and present tools for performing qPCR in a practical, simple, and efficient manner.