quantitative (real-time) PCR
/ qPCR /
Ordinary PCR can tell you whether a target is present, but it is bad at telling you how much was there to begin with, because the reaction runs out of steam and plateaus. Quantitative PCR fixes this by watching the copies appear in real time, as the reaction runs, and using how soon they appear to calculate the starting amount. It answers not just 'is it there?' but 'how much of it is there?'
qPCR adds a fluorescent reporter to the reaction — either a dye that glows when it binds double-stranded DNA, or a sequence-specific probe that lights up only when the target is copied. After each cycle the machine measures the fluorescence, which is proportional to how much product has formed. A sample that started with lots of target crosses a detectable brightness threshold after just a few cycles; a sample that started with very little needs many more cycles to get there. The cycle number at which the signal crosses that threshold is called the threshold cycle, or Ct, and it is the key measurement: the lower the Ct, the more target you started with. Crucially, this reads the amount during the exponential phase, before the plateau distorts things.
qPCR is the workhorse of measuring gene expression (how much mRNA a gene makes, after an RT step), viral load (how much virus is in a sample), and copy number. It is more quantitative than end-point PCR but it is relative, not absolute, unless you run a standard curve of known amounts — and like all PCR it amplifies contaminants, so a too-good signal can be a warning sign rather than good news.
Two patient samples are run for viral load. One crosses the fluorescence threshold at cycle 18, the other at cycle 30. Because each cycle roughly doubles the product, the difference of 12 cycles means the first sample started with about 2 to the 12th — roughly four thousand times — more virus than the second.
Earlier the signal appears (lower Ct), more target you started with.
qPCR is usually relative, not absolute — turning a Ct into a concrete copy number requires a standard curve of known amounts or a different method like digital PCR; and 'real-time PCR' is not the same as 'RT-PCR', though the two are often combined as RT-qPCR.