Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

A similar equation for the endogenous reference reaction is:CR T = R o × ( 1 + E R ) TR ,= K Rwhere:R T = threshold number of reference moleculesR o = initial number of reference moleculesE R = efficiency of reference amplificationC T, R = threshold cycle for reference amplificationK R = constantDividing X T by R T gives the following expression:X------ TR T=X o ( 1 E X ) C TX ,× +---------------------------------------------R o ( 1 E R ) C =TR ,× +K------- X= KK RThe exact values of X T and R T depend on a number of factors, including:• Reporter dye used in the probe• Sequence context effects on the fluorescence properties of the probe• Efficiency of probe cleavage• Purity of the probe• Setting of the fluorescence thresholdTherefore, the constant K does not have to be equal to one.Assuming efficiencies of the target and the reference are equivalent:E X = E R = E,X oC TX , – C------ TR ,× ( 1 + E ) = KR oor∆X N ( 1 E ) C T× + = Kwhere:X N∆C T= X o /R o , the normalized amount of target= C T, X - C T, R , the difference in threshold cycles for target and reference