Fam dye labeled taqman probes

For the quantification of mRNA expression, we applied the real-time fluorescence detection PCR method with FAM dyelabeled TaqMan probes (Applied Biosystems, USA). Values obtained for studied genes were normalized to the expression of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene as an endogenous control. The catalog numbers for the probes used are as follows: GPR30 -Hs01922715_s1, GAPDH -Hs99999905_m1. The real-time reaction mixture was prepared in a total volume of 10 µl and consisted of 0.5 µl cDNA, 5 µl TaqMan Gene Expression Master Mix, 0.5 µl TaqMan Gene Expression Assays and 4 µl RNA-free water and was performed as triplicate. The cDNA was amplified in Mastercycler Realplex (Eppendorf, Germany). Cycle parameters were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of sequential incubations at 95°C for 15 s and at 60°C for 1 min.
The fluorescent dye emission was a function of the cycle number. The initial amount of the template was evaluated as a Ct parameter. Ct value was the threshold cycle number at which PCR amplification reached a significant threshold. The number of the cycle was linearly correlated with logarithmic value of RNA quantity. The relative expression level normalized to GAPDH was calculated as 2^[-(Ct GPR30 -Ct GAPDH )] x1000.