PMC

Comparison of absolute and relative approaches to quantifying expression of c-fos in the eyes of wild-type and retinally degenerate (rd/rd cl) mice. (A) Absolute quantification, plotted as copies of c-fos normalised to copies of β-actin as determined from plasmid standard curves. (B) Relative quantitation expressed as c-fos R₀ normalised to β-actin R₀. Mean efficiency was derived from amplification plots without the use of a standard curve.

Determination of amplification efficiency from the linear phase of real-time PCR data. (A) Illustration of decline in amplification efficiency throughout a sample PCR reaction. Efficiency is plotted as rate of change in fluorescence on a cycle-to-cycle basis [(R_Ct/R_{Ct – 1}) – 1]. The cycle at which fluorescence equals M is indicated by a solid black line. Filled symbols represent a 10-fold range around this midpoint. (B) Illustration of the same sample data plotted as fluorescence on a logarithmic scale against cycle number. The midpoint of the detectable linear phase is shown as a broken black line. Filled symbols again represent the range around the midpoint used to calculate amplification efficiency.