Human dermal fibroblasts (Hu02) were transfected by pcDNA3.1(-)-VEGF vector. Following selecting fibroblast cells with hygromycin, recombinant cells were investigated in terms of VEGF expression by quantifying method. We used Real-Time PCR assay to quantitate gene expression of vascular endothelial growth factor from manipulated cells and represented VEGF overexpression.
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Reverse transcription was performed from 1 μg of total RNA transcribed to complementary DNA (cDNA) through 1 μL of random hexamer primer. The reactions were incubated at 70°C for 5 minutes. After that, 5X RT-buffer, dNTP, and RT-enzyme were added, and the mixtures were incubated at 42°C for 60 minutes and 70°C for 10 minutes. Reverse transcription was performed from 500 ng total RNA using the RT2 First Strand Kit (SA Biosciences). Quantitative real-time PCR was performed (Ampliqon, Denmark) with 40 cycles at 95 oC for 15 seconds and 60 oC for 60 seconds. The normalization and all the data analysis were performed according to RESR and Graph pad-Prism 8 software. For the normalization, it uses the housekeeping gene: β-Actin. Target gene signals normalized to housekeeping genes; 2^-deltaCt, where deltaCt = (Ct_Target − Ct_HKG)].