Hi! Murugadas A

As I understand you have 2 columns of Ct from the PCR machine soft.

First - gene of interest

Second - reference gene

Please, estimate the quality or your refernece. I mean standard deviation at least. If lower than 1 for all samples - great. If up to 2 - ok. if higher - it is not a good idea to use this gene like a reference.

Suppose that your reference gene is suitable.

If you do not know GOI and ref reaction effectiveness (standard curve) you can use 2^-dCt for data normalization.

for each sample:

a). Ct (goi) - Ct (reference) = dCt

b). 2^-dCt.

After that you will see the variation of your GOI.

Be careful if your reference is not good your data will be wrong and not relevant.

good luck

For relative gene expression analysis you can use geNorm program (http://medgen.ugent.be/~jvdesomp/genorm). Also dd(Ct) method can also be used.

For more details please refer the classical paper:

Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001; 29: e45.

Hello

Andrei S. Babenka is right and I dont think there is another option!