Key Factors That Affect Efficiency
Efficiency estimates can vary significantly if a rigorous measurement protocol is not followed
Measurements must be obtained over a broad dilution range (56 logs) and include replicates to decrease the effects of laboratory errors (e.g., pipetting accuracy)
Efficiency of a PCR reaction can be affected by substances known to inhibit PCR, such as heme and detergents. Users must ensure that cDNA templates are free of PCR inhibitors
In order to use the ΔΔCT method for relative quantitation as outlined in Applied Biosystems User Bulletin #23, the efficiency (EX) of the target amplification and the efficiency of the endogenous control amplification must be approximately equal (refer to references 1 and 3 for derivation of the ΔΔCT equations).
The results in this application note show that all of the TaqMan Gene Expression Assays have amplification efficiencies that are equivalent, because they all have an efficiency of one (EX = 1.0).
In this study, efficiency values were measured using the CT slope method. This method involves generating a dilution series of the target template and determining the CT value for each dilution. A plot of CT versus log cDNA concentration is constructed. With this method, the expected slope for a 10-fold dilution series of template DNA is -3.32, when EX = 1.0.
All TaqMan Gene Expression Assays have been designed by the Applied Biosystems genome-aided primer and probe design pipeline