Real-time Quantitative PCR

The increasing fluorescence signal plotted on the y-axis and the increasing thermocycle numbers along the x-axis generate a sigmoidal curve. The threshold cycle is defined as the cycle number when the fluorescent signal exceeds the detection threshold. During the following 4–8 cycles, the fluorescence typically approaches an exponential increase before approaching a plateau phase. During the exponential phase, the logarithm of the fluorescent signal along the y-axis and the cycle number along the x-axis generate a straight line that can be used for quantification. In this phase of the real-time PCR, the fractional threshold cycle number (C_t) is inversely related to the logarithm of the number of starting templates. Parallel PCR amplification of a dilution series of known amounts of the same template therefore allows the construction of a calibration curve from which the exact amount of starting template can be calculated. It must then be assumed that the amplification efficiency is equal for all samples and standards. One sign of acceptable quantification is that the slope of the calibration curve is between -3.3 and -3.4. More exact guidelines to quantification and quality control can be found in user bulletins to thermocycler software (e.g. ABI Prism User Bulletin No. 2, http://www.appliedbiosystems.com/).
 Absolute and Relative Quantifications
In order to achieve absolute quantification, a known amount of reference standard is required for the dilution series and calibration curve generation. Synthetic oligonucleotides, purified PCR fragments or plasmids or in vitro transcribed RNA can be quantified using absorption readings at a wavelength of 260nm (OD260). Dilution series of exactly known amounts of target then allow absolute quantification. Relative
quantification, relating the amount of template to an accepted standard, is often sufficient and this can be achieved using the calibration curve method or the comparative C_t method .