Real-time PCR is used to quantify starting amounts of nucleic acid template by analysing the amount of DNA produced during each cycle of PCR. It is a form of quantitative PCR or Q-PCR.
Real-time PCR is often preceded by reverse transcription to detect 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.
An excellent and fast way to select primers is with the free online-tool Primer3, currently in v0.3. Primer3Plus, a variation of Primer3 has qPCR settings. Or just apply the following or similar settings to Primer3:
- pair towards 3' end (often more specific, some cDNAs don't contain)
- pair separated by an exon-exon boundary (reduces genomic background) e.g. last exon & penultimate
- amplified region must be no biger than 200 bp; usally 60-150 bp
- GC content: 50-60%
- min length: 18, max length 24 (best: 20 nt)
- melting temperature: min 60, max 63, best 60
- max Tm difference: 10 (shouldn't be more than 1 in final pair)
- max 3' self complementary: 1
- max poly-x: 3
Verify by blasting the primers sequences. Target gene should come out with the lowerst E value. No other gene should be close. Also check whether possible isoforms will be detected by the candidate primer pair.
- 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.