The SmartFlare that we have chosen for this project is specific to vascular endothelial growth factor (VEGF) mRNA.
VEGF was selected as the target because it’s transcript becomes increased in a cell as part of a characterized response to hypoxia with the aid of hypoxia inducible factor-1 (HIF-1). Active HIF-1 is a heterodimer of α and β subunits, levels of HIF-1α are kept low by oxygen-dependent prolyl hydroxylases (PHDs). When a cell becomes hypoxic, PHD levels are reduced so more HIF-1α is present to dimerise with HIF-1β forming the active heterodimer that can act as the VEGF transcription factor.
Dimethyloxalylglycine (DMOG) is a PHD inhibitor, this means that it can mimic the hypoxic state of the cell to increase the levels of HIF-1α that leads to more VEGF in the cells, acting as a great positive control in our cell system (you can find plenty of in vivo and in vitro papers at PubMed).
In a quantitative PCR experiment, SK-N-AS cells that were treated for 1 or 3 days with 500nM DMOG showed a 13 and 34-fold increase of VEGF mRNA respectively.
Given our experimental setup, we will use cells treated with DMOG as a positive control, to ensure that there is a detectable level of VEGF mRNA within the cells for the Cy3-VEGF SmartFlare.