Relative quantification methods

Relative quantification methods provide information on the identity of peptides/proteins in a sample as well as their levels expressed in amounts relative to each other. Some of these methods rely on label-free strategies while others incorporate stable isotopes into one or more of the samples, allowing them to be combined and analyzed together. In both cases, the strength of the signal for each peptide is a reflection of the amount of peptide present in the sample, providing quantitative information. 

The third advantage of quantitative real-time RT-PCR is that it allows one to monitor the treatment effect in individual patients as a potential surrogate marker after adjuvant chemotherapy (K3) or immunotherapy (S4) against micrometastatic diseases. For the purpose of monitoring, a relative quantification method which is designed to determine exact, PCR efficiency-corrected mRNA concentration, normalized to a calibrator, might be desirable to overcome the inter-assay variation from run to run (SI3). This may make it possible to directly compare the mRNA values at different time points. 

One disadvantage of photon activation analysis is the comparatively poor detection power for several elements, e.g., sodium, vanadium, cobalt, the rare-earth elements. Thus, ultratrace determinations of these elements are barely possible using photon activation. As in other instrumental analytical techniques, photon activation analysis is a relative quantification method and hence needs reference materials with known compositions. (Reference materials are dealt with below.)... 

In the relative quantification method, a calibrator is utilized to measure the relative change of samples in gene expression. The calibrator can be an endogenous control, an exogenous control, or a reference gene (Dorak, 2006). 

Ikins, W. G., Kwak, H. S., Zink, G. S., and Jeon, I. J. (1988). A comparison of three relatively rapid methods for quantification of free fatty acids in cheese.. Food Sci. 53,1915-1916. 

Two methods are usually used for the evaluation of real-time PCR data, namely the standard curve method and the comparative threshold (Ct) method. The standard curve method relies on the use of purified cDNA pasmid standards, which will result in only a relative quantification. Other more specific standards can be used, e.g., in vitro transcribed RNA, which gives an absolute qantitation, however, this method is very labour intensive and not commonly used (Martell et al. 1999). The standard curve is included in each PCR run, and therefore provides a correction control for the PCR efficiency, making inter-assay comparisons easier. The comparative Ct method uses algorithms to calculate relative expression levels, compared to a calibrator (e.g., a control sample). A detailed description of the mathematics is given by Livak and Schmittgen (2001). After calculation the normalized expression... 

In the previous sections, comparative quantitative proteomics is discussed. This is aiming at relative quantification of protein levels in various cell states. In some applications, absolute rather than relative quantification of proteins is relevant, e.g., as an alternative to immunoaffinity methods for quantitative bioanalysis of protein dmgs. This requires different strategies. 

In general, two different strategies are possible for relative quantification (I) the relative standard curve method and (2) the comparative Ct method (AAC ). Depending on various factors, one method may be more appropriate than the other. The advantages of each method and factors to be considered are described below. 

A number of different methods to monitor the amount of methylimidazole left in a final ionic liquid are known. NMR spectroscopy is used by most academic groups but can have a detection limit of about 1-2 mol%. The photometric analysis described by Holbrey, Seddon and Wareing has the advantage of being a relatively quick method that can be performed with standard laboratory equipment [10], making it particularly suitable for monitoring the methylimidazole content during commercial ionic liquid synthesis. The method is based on the formation and colorimetric analysis of the intensely colored complex of 1-methylimidazole with copper (II) chloride but suffers from lack of precise quantification. 

As it has been shown above, the quantification of LCC linkages with traditional wet chemistry methods is limited mostly to relative quantification of carbohydrate sites linked to lignin [18,67-74]. 

As it has been shown earlier, the quantification of LCC linkages with traditional wet chemistry methods is limited mostly to relative quantification of carbohydrate sites linked to lignin. In contrast, our quantitative 2D NMR approach [21] allows for quantification of lignin sites involved in LCC linkages of different types. However, it does not provide information on the specific carbohydrate linkage sites yet. Therefore, a combination of quantitative NMR analysis and appropriate wet chemistry methods, such as a routine carbohydrate analysis, along with the methylation and DDQ oxidation degradation techniques, could be the best approach for comprehensive LCC analysis. 

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