Calculating Relative Expression??

[Wipawan]

I new at qRT-PCR technique. I have looked at the expression of resistant gene in bacteria after treating with antibiotic. I used 16srDNA as a endogenous control. I have two question about this?

1. Should I determined RNA amount before realtime PCR process (because my lab don't have nanodrop or spectrophotometer at 260 nm T_T)

2. I try to done qPCR without calibrate RNA. The results showed ct values of target gene in untreated is 22 and treated is 28, respectively while ct values of endogenous in untreated is 11 and treated is 23, respectively

I use 2–ΔΔCt for calculate relative expression

fold change = 2^-(ΔCt target - ΔCt calibration)

fold change = 2^-((28-23)-(22-11))
fold change = 2^-(-6)
= 64

I that mean the treated had over-expression than untreated 64 fold ????
My hypothesis is this gene might underexpressed after treating with antibiotic
But the result was opposite

Can anybody suggesting me ???