Vic labeled probe

Quantitative RT-PCR analysis was performed to determine the mRNA expression levels of C3 and GAPDH. The total RNA was harvested from the serum of all individuals using an RNeasy kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. Briefly, the PCR procedures were as follows: Pre-denaturation at 95°C for 30 sec, followed by 35 cycles of denaturation at 95°C for 15 sec and annealing at 56°-60°C for 30 sec. RT-PCR experiments were performed a minimum of three times.
RNA (1 μl) was reverse transcribed into cDNA using random primers in a Reverse Transcription II system (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. The mRNA expression levels of C3 and GAPDH were determined using quantitative PCR with an ABI Prism Sequence Detection system (Applied Biosystems Life Technologies, Foster City, CA, USA). The primers used are listed in Table I. An assay reagent containing premixed primers and a VIC-labeled probe (Applied Biosystems Life Technologies; cat no. 4310884E) was used to quantify the mRNA expression level of endogenous GAPDH. The relative levels of C3 transcripts were normalized against the amount of GAPDH mRNA for each sample.

BBB-ECs were grown to confluence on gelatin-coated 6-well plates (72 h) before inflammation with TNF/IFN-γ (100 U/ml) in the presence of rhEGFL7 (10 μg/ml) or vehicle. After 18 h, cells were harvested by scraping and quantitative PCR was performed as previously published^{20 (link)}. Relative gene expression levels were determined using primers and TaqMan® FAM^TM-labeled MGB probe for MCAM, VCAM-1, ICAM-1, and ALCAM and ribosomal 18 S (VIC®-labeled probe; Applied Biosystems). Gene-specific messenger RNA was normalized as compared to endogenous control 18 S.