CtNorm: Real time PCR cycle of threshold (Ct) normalization algorithm

In relative quantification with Real Time PCR (qRT-PCR,), accurate analysis requires equal amplification efficiency for both genes (Gene of interest and reference gene) and equal threshold values for all the samples. In this quantification method the expression level in treated samples will be calculated in comparison to the control group. We conducted the present study to design an algorithm for converting the data obtained from different runs containing identical standard samples into one run with the same amplification efficiency and threshold value. For this purpose, two formulas were designed; one to convert the amplification efficiency of the each run to 100%, and the other one for converting data from different runs into one run. Utilizing these two formulas, an algorithm was developed and named CtNorm. The online version of CtNorm algorithm is available at http://ctnorm.sums.ac.ir/. We used qRT-PCR technique to validate the accuracy of the designed algorithm for the normalization of four different human internal control genes. Normalizing the Ct values obtained from separate runs with the CtNorm algorithm has eliminated the differences and the average of the Ct values has become similar to the condition in which all the samples were amplified in a single run. The CtNorm algorithm could be utilized for equalizing the Ct values of several qRT-PCR runs with the same standard samples. The algorithm has also the ability to convert the amplification efficiency to 100% which is useful in absolute and relative quantification.