The objective of this study is to investigate the use of qRT-PCR as an early-stage bioassay to quantify biological activity. The action of VEGF165 on human umbilical vein endothelial cells (HUVECs) was selected as a model since we have previously studied dose-dependence and time courses for several late-stage responses for this system (production of IL-6, IL-8 and tissue factor, cellular proliferation and survival). Assay of VEGF activity and inhibitors underpins a variety of current drug development programs. HUVECs were cultured in 24-well plates and stimulated by incubation with a range of VEGF concentrations (0250 ng/ml) for 20, 45 or 90 minutes. To screen for a suitable target gene, mRNA levels for several genes (cfos, FosB, IL-6, IL-8 and tissue factor) were measured using an automated qRT-PCR platform (Rotorgene 6000, Corbett Research). A dose-dependent increase in the levels of all these mRNA species was detected after 45 minutes, with the highest increases in cfos mRNA levels at 20 minutes. Increases over control (zero dose VEGF) mRNA levels ranged from 3-fold (IL-6) to 17-fold (tissue factor) after 45 minutes. Maximum increases in mRNA levels were obtained with 15.6 or 62.5 ng/ml VEGF, i.e. over a similar dose range to the late-stage responses. With the exception of tissue factor, mRNA levels of all gene targets decreased markedly after 90 minutes exposure to VEGF.
These results demonstrate that measurement of mRNA levels could provide an early-stage bioassay for VEGF and possibly other biologically active materials. The shorter incubation time compared with the late-stage assays (several hours or days) removes the need for sterile handling of cells and samples during the assay. The later stages of the assay require a real time delay, but are fully automated. We are extending the assessment to further analytes and other culture formats.