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Amplification Efficiency of TaqMan Gene Expression Assays

nvariant endogenous control. The results obtained for GAPDH are shown in Figure 5. The fold change results obtained using the ΔΔCT method showed the best correlation to the expected fold change (slope = 0.94; expected slope = 1.0).

The ΔΔCT method assumes that the amplification efficiency for both assays (GAPDH and NHT) is very similar and close to 100%. The fold change calculated using measured efficiency values (GAPDH assay efficiency = 97.5% and NHT = 94%) had a poor correlation to the expected fold change (slope = 0.78; expected slope = 1.0). The data points to the potential hazard of using pre-determined efficiency values to do relative quantitation calculations. If the efficiency determinations used in the equations are inaccurate, then the error is propagated into the relative quantitation calculations, i.e., the more biologically relevant results.

Conclusions and Recommendations

Our results show that when you use TaqMan Gene Expression Assays, calculated fold change values correlate better to expected fold change values when the nnCT method for relative quantitation is used. This is because all of our assay products meet the assumptions necessary to use this simplified equation. In other words, the amplification efficiency of all TaqMan Gene Expression Assays are equivalent to any other target assay because they all have amplification efficiencies very close to one.

Therefore, when using TaqMan Gene Expression Assays, it is not necessary to measure efficiency. We have extensively tested our design parameters and are confident that the resulting assays will have 100% efficiency (+/-10%) when measured over a 6-log dilution range, in samples that are free of PCR inhibitors. Determination of amplification efficiency (EX) using a template dilution series can generate highly variable results depending on the conditions
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