What’s the significance of ROX reference dye in real time PCR (qPCR)?

ROX reference dye is essential to the accuracy and reproducibility of qPCR reactions performed on ROX-dependent PCR cyclers (e.g., the Applied Biosystems® instruments). When added to the qPCR master mix, the ROX Reference Dye is designed to normalize the fluorescent signal of qPCR reporter dyes or hybridization probes and account for non-PCR-related fluorescence signal variations, including pipetting errors, bubbles, condensation, evaporative loss, and instrument variation. Since the ROX Reference Dye does not interfere with the qPCR reaction and has an easily discernable red emission spectrum, it provides an excellent baseline in multiplex qPCR assays.