Quantitative analysis amplification assays

Because the templates compete for amplification and, in the case of reverse transcription PCR (RT-PCR), also for reverse transcription, any variable affecting amplification has the same effect on both. Thus, the ratio of PCR products reflects the ratio of the initial amounts of the two templates as demonstrated by the function C/W=C (l+ )"/Wi(l+ )n, where Cand Ware the amounts of competitor and wild-type product, respectively, and C and W are the initial amounts of competitor and wild-type template, respectively, (Clementi etal., 1993). From this linear relationship, it could be concluded that a single concentration of competitor could be sufficient for quantitating unknown amounts of wild-type templates. However, in practice, the precise analysis of two template species in very different amounts has proved difficult and cPCRs using three to four competitor concentrations within the expected range of wild-type template concentrations are usually performed. In a recent study of different standardization concepts in quantitative RT-PCR assays, coamplification on a single concentration of a competitor with wild-type template was comparable to using multiple competitor concentrations and was much easier to perform (Haberhausen et al, 1998). 

The methods outlined below include protocols for direct and indirect immunofluorescence staining, that can be adapted easily for the cell type of interest as indicated in the relevant notes. The principal approaches to flow cytometric analysis, standardization and calibration are then given, followed by two more detailed protocols illustrating quantitation using direct immunofluorescence, and a competitive binding assay, which demonstrates the application of linear amplification of fluorescence. 

In quantitative mass spectrometry, the signal intensity depends not only on the amount of sample, but also on a number of other variables such as the ionization yield, focusing of the ion beam, and the amplification factor of the detector. As it is very difficult to keep these parameters constant over the whole period of analysis, nearly all quantitative applications of MS are based on a comparison of the ion current obtained from the component of interest, with the ion current obtained from a standard. In quantitative SIM this can be accomplished either by the continuous admission of a reference sample at a constant rate, concurrently with the sample under investigation, or by the use of an internal standard (IS) which is added to the sample prior to MS analysis (Halpern, 1981). The choice of this IS is of primary importance in the design of a new assay and was subject to some controversy in the late 1970s (Claeys et al., 1977 Lee and Millard, 1975 Millard, 1978b Self, 1979). Ideally, an IS should compensate for all possible losses during sample isolation, purification, derivatization, and separation steps and at the same time minimize variances due to the measurement process. In practice, the... 

Immunoreagents are well suited for quantitation of amplified DNA. However, the exponential nature of PCR amplification makes it difficult to extrapolate from the amount of amplified DNA to the amount of starting material. The most reliable quantitative PCR methods involve real-time assay of amplified DNA during the exponential phase of the amplification reaction. Because the analyte of interest is usually the DNA template, not the amplification product, immunochemical methods at their present stage of development are generally best applied to an unamplified template where concentrations permit direct analysis or for qualitative assays. 

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