Quantitative PCR (qPCR) is an increasingly popular method of nucleic acid quantification. With traditional endpoint PCR, the amount of amplified product is typically determined only after the final amplification cycle is completed. This is usually after the plateau phase of amplification, where reaction components have become depleted, and thus this estimate may not be a true representation of the amount of starting material. With qPCR, the amount of amplified product is measured at the end of each cycle of amplification or in real time during the exponential phase of amplification. After each cycle of amplification, amplification product accumulates and eventually reaches a threshold, where it can be detected above background. This point, or cycle, in the reaction is referred to as the Quantification Cycle, or Cq, and is often set at a signal that is ten standard deviations above the background signal.
The amount of amplified product is measured by fluorescence, either through the use of DNA-binding fluorescent dyes (e.g., BRYT Green®, SYBR® Green), fluorescently labeled DNA probes (e.g., Taqman® Assays, Molecular Beacons), or fluorescently labeled DNA primers (e.g., Plexor® Systems). Quantification can be relative, such that the quantification cycle value (Cq) of the gene of interest is compared to that of a reference gene, or absolute, such that a standard of known DNA amount is analyzed along with the samples of interest to obtain a defined amount of DNA in the starting material. Theoretically, each cycle results in the doubling of the product (at 100% efficiency), meaning a difference of 1 Cq corresponds to a twofold difference in starting material, while a difference of 3.3 Cq corresponds to a tenfold difference in starting material.
RT-qPCR can be used to measure the amount of a specific RNA target in a sample by incorporating a reverse transcription (RT) step followed by qPCR. Primer design is crucial for successful RT-qPCR. The use of RNA-specific primers that flank an intron of the target sequence are recommended for RNA (cDNA) detection, whereas primers within an intron can be used to detect DNA contamination in an RNA sample. In addition, control reactions in which no reverse transcriptase is added to the RT reaction are recommended for analyzing DNA contamination. RT-qPCR is extremely sensitive, and under optimal reaction conditions, a single copy of a target sequence can be detected. Amplicon sizes are typically 100–200bp to ensure good amplification efficiency. Primers that generate amplicons of various lengths can be used to determine RNA integrity. As RNA becomes increasingly degraded, it becomes more difficult to successfully and consistently amplify larger amplicons. Thus, for degraded RNA, smaller amplicon sizes are recommended.
Labeling separate primer sets with different fluorophores allows the user to analyze multiple targets in a single reaction and analyze samples for the presence of PCR inhibitors. To detect PCR inhibitors in the RNA sample, the samples are added to a reaction that contains a control DNA template and primers. Any delay in Cq value compared to the Cq value of the control DNA alone may indicate the presence of PCR contaminants that could affect downstream applications (e.g., Plexor® or Taqman® inhibitor assay). While often the downstream application for RNA analysis, RT-qPCR can be used to qualify RNA for other downstream applications as well. One disadvantage to qPCR and RT-qPCR is cost because real-time analysis often requires the use of specialized reagents and instrumentation.