Methods

Theoretical Notes

 Experimental Design

Data Analysis

Primer Design

 Comparative CT Method:

Please refer to relative standard curve method for background information prior to reading comparative CT method. Comparative CT method is essentially an abbreviated version of the relative standard curve method.

Using the comparative CT method utilizes the assumption that the primer efficiencies are relatively similar. As seen in the theory section, the primer efficiencies cancel if they are the same. Thus, in assuming this, the standard curve can be omitted from the assay.

As derived in the theory section, the

 

(1)

where the error is computed as the coefficient of variation (standard deviation normalized to the mean)

(1a)

The error of the control sample does not enter the equation since the control sample is taken as an arbitrary constant. As such, the reference sample could be any endogeneous control.

where CT,X is the threshold cycle of the gene of interest and CT,R is the threshold cycle of the endogenous reference gene (e.g. GAPDH). Test refers to the test cDNA sample and Control refers to the control cDNA sample.

Prior to utilizing this method, it is necessary that the primer efficiencies are shown to be relatively similar by experimentation. Primer efficiency is defined as in equation 2.

(2)

where slope is the slope of the standard curve plot.

To show that the primer efficiencies are similar, simply run a standard curve for both the endogeneous control primer set and gene of interest primer set as outlined in the relative standard curve method. If the primer efficiencies for both genes are similar, then you can proceed with the comparative CT method. The comparative CT method offers the advantage of being able to omit the standard curve samples from the experiment, thus increasing your throughput allowing you to simultaneously screen more cDNA samples in one experiment.

The difference in the primer efficiency between the gene of interest primer set and endogeneous control primer set should not exceed 5%. Figure 1 shows an instance where the comparative CT method can be utilized because the standard curves are superimposable and essentially identical primer efficiencies.

 

 

Where 11881 is the gene of interest. The efficiencies for GAPDH and 11881 were 98.4% and 97.3% respectively. Thus, the difference is less than 5%.

 

Once the primer efficiencies proven to be similar by experimentation, you can proceed with the comparative CT method. Table 1 shows the raw data used to compare the expression ratio of 11881 in two cDNA samples. For this purpose, we will ignore the standard curve samples for our calculations.

Table 1: CT data obtained for Gene 11881.

A sample calculation for computing the expression ratio

Table 2 shows the values for the expression ratios computed using the comparative CT method. As seen from the data, 11881 is virtually not present in our test sample.

Table 2: Expression ratios computed utilizing the comparative CT method.

Taking a step further and recomputing our data using the relative standard curve method, we can see that the comparative CT method and relative standard curve method compute very similar expression ratios. As stated earlier, under the validation that the primer efficiencies are similar, the comparative CT method can be utilized as a substitute to the relative standard curve method.


Table 3: Expression ratios computed utilizing the relative standard curve method.

 

Methods

Theoretical Notes

 Experimental Design

Data Analysis

Primer Design

RRC Core Genomics Facility
University of Illinois at Chicago
2003