ANOVA (analysis of variance) can be used to compare differences in amplification efficiencies between genes.
Often abbreviated to Q-PCR, this method is sometimes also referred to as real-time PCR or depending on the application, quantitative reverse-transcriptase PCR (both of which are abbreviated to RT-PCR, which can be rather confusing).
One of the first is known as the ΔΔ method requires that the reference gene is amplified at a similar efficiency to that of the target gene and that it is not dramatically affected by the experimental treatment.
An initial set of experiments should be conducted to test whether or not these are true.
A 100% efficient reaction will have a slope of −3.32.
During exponential amplification, a 100% efficient reaction will double every cycle and will produce a 10-fold increase in PCR product every 3.32 cycles.method for measuring relative gene expression, the following equation can be used to determine an initial amount of fluorescence at zero time (when the temperature is shifted, when cells are infected, when cells are exposed to a drug, etc.).