Dilution method

The amplification rate is calculated on the basis of a linear regression slope of a dilution row (Figure 3.3). Efficiency (E) can be determined based on eq. 9 (Higuchi et al., 1993; Rasmussen, 2001). But the real-time PCR efficiency should be evaluated sample-by-sample, which is quite laborious and costly, wastes template, and takes time if the dilution method is used. Alternatively, the pool of all sample RNAs can be used to accumulate all possible 'positive and negative impacts' on kinetic PCR efficiency. Applying the dilution method, usually the efficiency varies in a range of E = 1.60 to values over 2 (Figure 3.3) (Souaze et al., 1996).

Typically, the relationship between CP and the logarithm of the starting copy number of the target sequence should remain linear for up to five orders of magnitude in the calibration curve as well as in the native sample

35 n

ng cDNA vs. gene 1; slope = -3.108; E = 2.09 ng cDNA vs. gene 2; slope = -2.986; E = 2.16 ng cDNA vs. reference; slope = -3.342; E = 1.99 regressions

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Figure 3.3

On the basis of a dilution row the real-time efficiency is calculated according to eq. 9 (Higuchi et al., 1993; Rasmussen, 2001).

RNA (Muller et al., 2002). The advantage of the dilution method is that it is highly reproducible and constant within one transcript and tissue. The disadvantage of this approach is the high efficiencies, often higher than two (E > 2.0), which is practically impossible on the basis of the PCR amplification theory. This indicates that this efficiency estimation is more or less not the best one and it will overestimate the 'real' amplification efficiency.

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