What we hope to have presented here is a coherent flow through the considerations when designing an mtDNA real-time PCR assay. Many of these considerations are no different to when designing any real-time assay, nuclear or mitochondrial, and where this is the case we have directed the investigator to the relevant chapter. As a large amount of mitochondrial research is carried out at the level of the individual cell, we feel that it is these considerations that should strongly influence the design and

Figure 11.4

Effect of final Magnesium Chloride concentration of real-time PCR efficiency. Each graph shows a 1:10 serial dilution series, amplified for a region of nD1. Inset on each graph is the standard curve at that magnesium chloride concentration showing the equation for the line, the R2 value and the PCR efficiency. A. 1.5 mM; B. 2.5 mM; C. 3.0 mM; D. 3.5 mM; E. 4.0 mM; F. 4.5 mM. Improved reaction efficiency can be seen as the magnesium chloride concentration increases.

optimization of a real-time assay and have focused the attention of this chapter in that direction.

The variability in the real-time PCR machines and in the different manufacturers' reagents, together with the numerous different targets make it impossible to generate a generic protocol for an mtDNA real-time assay. We have covered what we believe to be the main considerations when working in the mtDNA field and feel it is sufficient to say that any real-time assay has the potential to generate strong data if correctly optimized. We would advise strongly against trying to cut corners and costs by minimizing the optimization and replicate analysis. Optimization and validation is essential to be able to draw conclusions from real-time data and should be carried out in accordance with guidelines laid out in the other chapters of this book. The conditions found to work for one assay will not necessarily work well for all.

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