Q Pcrefficiency Calculator

Amplification efficiency describes how close a PCR reaction comes to doubling its target every cycle. Because product accumulates exponentially, the quantification cycle Cq falls linearly as the log of starting template rises, and the slope of that standard curve carries the efficiency: the per-cycle amplification factor equals 10 raised to the power of minus one over the slope. Perfect doubling corresponds to a factor of 2 and a slope of -3.322, since log10(2) = 0.301. Practitioners run a dilution series of at least five points spanning three or more orders of magnitude, require a coefficient of determination above 0.98, and accept efficiencies of 90 to 110 percent, which correspond to slopes between about -3.6 and -3.1. Efficiency matters because relative quantification is exponential in Cq. The Livak or delta-delta Cq method assumes the target and reference assays both amplify with a factor of exactly 2, so a 10 percent efficiency error compounds across a Cq difference of 5 into roughly a 25 percent error in reported fold change. The Pfaffl method removes that assumption by raising each assay's measured efficiency to its own delta Cq. Apparent efficiencies above 110 percent usually signal pipetting error in the dilution series, inhibitors that suppress only the concentrated standards, or non-specific product; values below 90 percent point to primer secondary structure, an amplicon longer than about 200 base pairs, suboptimal annealing temperature, or carryover inhibitors such as humic acids, heparin, or haem. Cq itself shifts with baseline and threshold settings, so efficiency should be compared only within a consistent analysis method.

PCR was conceived by Kary Mullis at Cetus Corporation in 1983 and published in 1985 and 1986, work that earned him a share of the 1993 Nobel Prize in Chemistry. It was originally an endpoint technique: product was run on a gel after cycling, which made quantification crude because reactions plateau as reagents deplete. Real-time monitoring arrived in 1992 and 1993, when Russell Higuchi and colleagues at Roche Molecular Systems added ethidium bromide to reactions and recorded fluorescence with a video camera during thermal cycling, showing that the cycle at which signal crosses a threshold is proportional to the log of input template. Sequence-specific detection followed from the 5-prime nuclease assay described by Pamela Holland and colleagues in 1991 and turned into the dual-labelled TaqMan probe by Livak and co-workers in 1995; the ABI Prism 7700 instrument commercialized the approach in 1996. The two dominant analysis formulas were both published in 2001. Kenneth Livak and Thomas Schmittgen described the 2 to the minus delta-delta Ct method in Methods, together with the validation experiment that checks whether target and reference efficiencies are close enough to use it. Michael Pfaffl published his efficiency-corrected relative expression model in Nucleic Acids Research the same year. Widespread inconsistency in how laboratories reported these numbers led Stephen Bustin and a group of co-authors to publish the MIQE guidelines in Clinical Chemistry in 2009, which standardized the term Cq and made reporting of amplification efficiency, linear dynamic range, and reference gene validation an expectation for publication.