Real-time quantitative RT-PCR was performed in a real-time thermocycler (LightCycler; (Roche Diagnostics Canada, Montreal, Quebec, Canada) using the SYBR Green I detection platform (Molecular Probes, Eugene, OR). SYBR Green I is a double-stranded (ds)DNA–binding dye that allows kinetic quantification of PCR products. PCR reactions were performed in glass microcapillaries (Roche Diagnostics) in a total volume of 20 μL. The reaction mixture consisted of 2.5 μL of 10× PCR buffer (Invitrogen Inc.), 1.25 μL of 5 mM dNTP, 1.2 μL of 50 mM MgCl
2 (1.6 μL for rat TGF-β1 and ET-1), 1 μL of each forward and reverse 10 μM primer
(Table 1) ,
28 29 0.5 μL of 5 U/μL platinum DNA
Taq polymerase, 0.75 μL of 10× SYBR Green I, 10.8 μL H
2O, and 1 μL cDNA template. For human ET-1 and TGF-β1, we designed minor groove-binding probes (
Taqman; Applied Biosystems, Foster City, CA) on computer (Primer Express, ver. 2.0; Applied Biosystems) to avoid signal acquisition from nonspecific amplification products. These probes are modified at the 5′ end by the addition of 6-carboxyfluorescein (FAM) and at the 3′ end by the addition of a nonfluorescent quencher. As elongation proceeds, FAM is cleaved by the exonuclease activity of DNA
Taq polymerase and an increase in reporter fluorescence emission takes place. The reporter dye (FAM,
Taqman, Applied Biosystems) exhibits excitation and emission in the same range as SYBR I, which allows detection with the same detector channel. The PCR reaction mixture for human ET-1 and TGF-β1 was the same, with the exception of 0.75 μL of 15 μM probes instead of SYBR I.
During PCR reactions, an additional step was incorporated after the extension phase, to minimize signal interference from primer dimers and nonspecific amplification products.
30 This signal acquisition phase was empirically determined by obtaining the melting temperature (T
m) of all products by melting curve analysis (MCA). During MCA, post-PCR products were allowed to anneal at 60°C before the temperature was increased at 0.10°C per second. The signal acquisition step was set to 2°C to 3°C below the T
m of the specific amplification product
(Table 2) .
mRNA levels were quantified using the standard curve method. Standard curves for all genes were constructed by using serially diluted standard template. Crossing point (Cp; log-linear phase) was measured for all samples and used to compute mRNA levels from the standard curves. The data were normalized to a housekeeping gene (18S rRNA) to account for differences in reverse transcription efficiencies and the amount of template in the reaction mixtures.