(primer design section moved to qRT-PCR)
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Real-time PCR is often preceded by reverse transcription to quantifiy RNA via their cDNA. In fact, mRNA quantification is one of the most frequent uses of real-time PCR. The sub-technique is sometimes called qRT-PCR for quantitative reverse transcription PCR.
Amplification of DNA is exponential in the early and middle cycles of a PCR (i.e. it is linear on a logarithmic scale). This property can be exploited to infer the starting amount of PCR template (see diagram in Hunt tutorial). During the exponential or log phase each copy of DNA is being amplified, and thus can be a better measure than in endpoint PCR, where reagents such a nucleotides may become exhausted and result in inefficient amplification, resulting in inaccurate quantification of the gene of interest.
Real-time PCR is more precise than previously used reverse transcription PCR (RT PCR) because the generation of product is continuously monitored during the PCR run (this is where the term "real time" comes in), rather than at the end of a PCR reaction ("endpoint" PCR).
Generation of product is detected in one of two ways . First, the amount of double stranded DNA in the tube can be measured using fluorescent dyes which intercalcate double-stranded DNA (like the DNA binding dye SYBR Green I). The intensity of fluorescence is proportional to the quantity of DNA present in the reaction. Second, the amount of PCR product can be measured by monitoring the hybridization of a set concentration of fluorescently labeled probe oligonucleotide. The oligo probe provide selectivity and only monitors the concentration of PCR product with a particular sequence. In contrast, SYBR green I will bind even nonspecific PCR products.
- When using SYBR Green I to measure DNA concentration, it is important to run the PCR product out on a gel to verify that there is only a single amplification product .
- When using SYBR Green I, the amount of fluorescence in a PCR product depends on the length and base composition of the product . So it is not possible to compare the concentrations of two different templates without having control templates of known concentration for each target DNA region.
- PCR, PCR techniques, Q-PCR and qRT-PCR on OpenWetWare.
- An excellent, detailed Q-PCR tutorial by Margaret and Richard Hunt, University of South Carolina
- Wikipedia entry on Q-PCR
- The venerable qpcrlistserv. Anyone doing qPCR should be subscribed to this list.