Protocol: Quantitative PCR for
Determination of Angiogenic Factors

Methods of Quantification

Standard curves are necessary for both absolute and relative quantification. When generating standard curves, different concentrations of DNA (typically five) should be used to generate a standard curve that will bracket the concentration of the unknown. Each concentration should be run in duplicate.
Reagents and kits for quantitative PCR may be used to quantify target DNA using either absolute or relative quantification. Absolute quantification techniques are used to determine the amount of target DNA in the initial sample, while relative quantification determines the ratio between the amount of target DNA and a reference amplicon. The ideal reference amplicon would have invariant, constitutive expression. In practice, a housekeeping gene is typically chosen for this function, but there are other reference choices which better adhere to the above requirements.3
Absolute quantification uses external standards to determine the absolute amount of target nucleic acid. To remove the differences in quantification due to annealing, the primer binding sites of the external standards must be the same as those in the target sequence. The ideal external standard contains sequences that are the same as or which vary only slightly from the target sequence. Equivalent amplification efficiencies between the target and external standard are necessary for absolute quantification. Once a suitable construct or amplicon is identified, a standard curve of external standard dilutions is generated and used to determine the concentrations of unknown target samples.
Relative quantification calculates the ratio between the amount of target template and a reference template in a sample. Since this method measures the amount of target relative to a presumably invariant control, relative qPCR is most often used to measure genetic polymorphism differences, for instance, between tissues or between healthy and diseased samples. The advantage of this technique is that using an internal standard can minimize variations in sample preparation and handling.
The accuracy of relative quantification depends on the appropriate choice of a reference template for standards. Variability of the standard will influence the results and so it is most important that standards be appropriate.3 Some researchers choose not to run a standard curve and report target quantities as a fraction of the reference, a technique termed comparative quantitation. Alternatively, one may assume that the difference in amplification efficiencies of target and reference is negligible and quantify the target based solely on the standard curve determined for the reference sequence. Finally, in the most accurate of the relative quantification techniques, the amplification efficiencies of both the reference and target are measured, and a correction factor is determined. This process, termed normalization,3 requires a sample containing known concentrations of both target and reference and the generation of two standard curves.
The PCR efficiency between a reference sample and a target sample is determined by preparing a dilution series for each target. The CT values from either the reference or target is then subtracted from the other. The difference in CT values is then plotted against the log of the template amount. If the resulting slope of the straight line is less than ±0.1, the amplification efficiencies are similar.
  1. Mocellin, S., et al., Quantitative real-time PCR in cancer research. Arch. Immunol. Ther. Exp. (Warsz), 51, 301-13 (2003).
  2. Bernard, P.S. and Wittwer, C.T., Real-time PCR technology for cancer diagnostics. Clin. Chem., 48, 1178-85 (2002).
  3. Bustin, S.A., Quantification of mRNA using real time reverse transcription PCR (RT-PCR): trends and problems, J. Mol. Endocrinol. 29, 23-9 (2002).
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