Retinal VEGF mRNA measured by SYBR Green I fluorescence: A versatile approach to quantitative PCR

PURPOSE: To determine whether continuous monitoring of SYBR Green I fluores cence provides a reliable and flexible method of quantitative RT-PCR. Our a ims were (i) to test whether SYBR Green I analysis could quantify a wide ra nge of known VEGF template concentrations, (ii) to apply this method in an experimental model, and (iii) to determine whether 20 existing primer pairs could be used to quantify their cognate mRNAs. METHODS: Real-time quantitative PCR was performed using a LightCycler rapid thermal cycler (Roche). Retinal VEGF mRNA levels were measured in a murine model of oxygen-induced retinopathy during vaso-obliterative and hypoxic p hases. RESULTS: This technique was able to detect as few as 10 control template co pies, with quantitative data available routinely for 1000 or more copies. T he levels of retinal VEGF mRNA expression followed the hypoxia-induced patt ern determined previously by conventional methods. All gene-specific primer pairs which amplify a specific product by conventional PCR were successful ly converted to SYBR Green analysis, including those for housekeeping genes glyceraldehyde phosphate dehydrogenase (GAPDH), cyclophilin, and acidic ri bosomal phosphoprotein PO (ARP/36B4) and for 28S rRNA. In each case melting curve analysis and agarose gel electrophoresis confirmed the specificity o f the amplification product. CONCLUSIONS: The sequence-independent detection of DNA with SYBR Green I me ans that it can be used to quantify the amplification of any cDNA using gen e-specific primers. This rapid and flexible method is ideally suited for re searchers in vision science wishing to quantify mRNAs from many different g enes because it does not require investment in gene-specific hybridization probes.