Hello Reshmi

with that normalised Ct value I.e Ct of GOI - Ct for housekeeper for both treated (test) and untreated (control = calibrator) you need to take those 2 delta Ct values and subtract from one another I.e (Ct target gene - Ct ref housekeeping gene: treated sample) - ( Ct  target gene - Ct ref gene: untreated control). 

The resulting delta delta Ct value 'x' should then be used as an exponent to the power of 2, I.e 2^{x}

This will give you the relative expression of the target gene in your treated sample versus your untreated control. The method was developed by Kenneth Livak and is explained in detail in the seminal paper below: 

It is this relative expression value you should subject to a Wilcox t test to evaluate significant difference in expression or alternatively statistical null difference in expression

on Monday when I return to work I can if required send you an excel spreadsheet with a worked example of this method plus other background clarifying materials 

As promised

Thanks Laurence.

Just to clarify to calculate the delta Ct of the reference gene do I use the individual reference gene (i.e. GAPDH) or the mean of the reference genes (i.e. GAPDH and Actin-B)?