Determining amplification efficiencies

The amplification efficiency of 2 genes (target A and target B) can be compared by preparing a dilution series for both genes from a reference RNA or cDNA sample. Each dilution series is then amplified in real-time one-step or two-step RT-PCR and the CT values obtained are used to construct standard curves for target A and target B. The amplification efficiency (E) for each target can be calculated according to the following equation:

E = 10(–1/S) – 1 (where S = slope of the standard curve)

To compare the amplification efficiencies of the 2 target sequences, the CT values of target A are subtracted from the CT values of target B. The difference in CT values is then plotted against the logarithm of the template amount (see figure Efficiency comparison). If the slope of the resulting straight line is <0.1, amplification efficiencies are comparable.

Efficiency comparison

Amplification efficiencies of the target gene and the endogenous reference gene are usually different since efficiency of primer annealing, GC-content of the sequences to be amplified, and PCR product size usually vary between the 2 genes. In this case, a standard curve needs to be prepared for the target gene as well as for the endogenous reference gene, for example, using total RNA prepared from a reference cell line (calibrator or reference sample).

Due to differences in PCR efficiency, the resulting standard curves will not be parallel and the differences in CT values of the target and the reference will not be constant when the template amounts are varied (see figure Different PCR efficiencies).

Different PCR efficiencies

Guidelines for relative quantification with different amplification efficiencies:

• Choose an appropriate endogenous reference gene whose expression level does not change under the experimental conditions or between different tissues.
• Prepare a dilution series (e.g., 5-fold or 10-fold dilutions) of a cDNA or RNA control sample to construct standard curves for the target and reference.
• Perform real-time PCR/RT-PCR.
• Determine the CT values for the standards and the samples of interest
• Construct standard curves for both the target and reference by plotting CT values (Y-axis) against the log of template amount or dilution (X-axis).
• Calculate the amount of target and reference in the samples of interest using their CT values and the corresponding standard curve.
• To calculate the normalized amount of target, divide the amount of target by the amount of reference (if replicate reactions were performed, use the average value).
• Define the calibrator sample and compare the relative expression level of the target gene in the samples of interest by dividing the normalized target amounts by the value of the calibrator.

If the amplification efficiencies of the target gene and the endogenous reference gene are comparable, one standard curve for the reference gene is sufficient. The differences in CT values of the target and the reference will be constant when the amounts of template are varied (see figure Same PCR efficiencies). The amounts of target and reference in an unknown sample are calculated by comparing the CT values with the standard curve for the reference gene.

Same PCR efficiencies

Guidelines for relative quantification with comparable amplification efficiencies:

• Choose an appropriate endogenous reference gene whose expression level does not change under the experimental conditions or between different tissues.
• Prepare a dilution series (e.g., 5-fold or 10-fold dilutions) of a cDNA or RNA control sample to construct a standard curve for the endogenous reference gene only.
• Perform real-time PCR/RT-PCR.
• Determine the CT values for the standards and the samples of interest
• Construct a standard curve for the endogenous reference gene by plotting CT values (Y-axis) against the log of template amount or dilution (X-axis).
• Calculate the amount of target and reference in the samples of interest using their CT values and the standard curve.
• To calculate the normalized amount of target, divide the amount of target by the amount of reference (if replicate reactions were performed, use the average value).
• Define the calibrator sample and compare the relative expression level of the target gene in the samples of interest by dividing the normalized target amounts by the value of the calibrator.

An alternative approach is the comparative or ΔΔCT method, which relies on direct comparison of CT values. The preparation of standard curves is only required to determine the amplification efficiencies of the target and endogenous reference genes in an initial experiment. In all subsequent experiments, no standard curve is required for quantification of the target sequence. If amplification efficiencies are comparable, amounts of target are simply calculated by using CT values as described below.

First of all, the ΔCT value for each sample is determined by calculating the difference between the CT value of the target gene and the CT value of the endogenous reference gene. This is determined for each unknown sample as well as for the calibrator sample

• ΔCT (sample) = CT target gene – CT reference gene
• ΔCT (calibrator) = CT target gene – CT reference gene

Next, the ΔΔCT value for each sample is determined by subtracting the ΔCT value of the calibrator from the ΔCT value of the sample.

• ΔΔCT = ΔCT (sample) – ΔCT (calibrator)

If the PCR efficiencies of the target gene and endogenous reference gene are comparable, the normalized level of target gene expression is calculated by using the formula:

• Normalized target gene expression level in sample = 2–ΔΔCT

However, if the PCR efficiency is not the same between the target gene and endogenous reference gene, this method of quantification may lead to inaccurate estimation of gene expression levels.

The error is a function of the PCR efficiency and the cycle number and can be calculated according to the formula:

• Error (%) = [(2n/(1+E)n) x 100)] – 100 (where E = efficiency of PCR; n = cycle number)

Therefore, if the PCR efficiency is only 0.9 instead of 1.0, the resulting error at a threshold cycle of 25 will be 261%. The calculated expression level will be 3.6-fold less than the actual value.

Tip: The ΔΔCT method should only be chosen if the PCR efficiency of the target gene and endogenous reference gene are the same, or if the difference in expression levels is sufficiently high to tolerate the resulting error. However, errors can be corrected by using efficiency-corrected calculation programs, such as the Relative Expression Software Tool (REST; see reference 5).

Guidelines for relative quantification using ΔΔCT method:

• Perform a validation experiment to determine the PCR efficiency for the target and reference (see Determining amplification efficiencies).
• Perform real-time RT-PCR for the target and reference with RNA derived from different samples.
• Determine the ΔCT value by subtracting the endogenous reference gene CT value from the target gene CT value for each sample.
• Define the calibrator sample and determine the ΔΔCT value by subtracting the calibrator ΔCT value from the ΔCT value of each sample.
• Calculate the normalized level of target expression relative to the calibrator by using the formula 2–ΔΔCT.