One-step NZYSpeedy RT-qPCR Green kit, ROX is an optimized and highly efficient reaction mixture developed for first-strand cDNA synthesis and subsequent real-time PCR in a single tube. The kit includes a One-step NZYSpeedy qPCR Green master mix, ROX developed for fast PCR, provided as a 2× reaction mixture, that contains all components necessary for real-time PCR, including a green intercalating dye, dNTPs, stabilizers and enhancers. In addition, a separate NZYRT mix which comprises a balanced mixture of both Reverse transcriptase and Ribonuclease Inhibitor is also provided. This kit enables fast and highly reproducible procedures on the most common real-time PCR apparatus from either total RNA or mRNA. It is optimized for intercalating green dye detection on different instruments. The latest developments in PCR enhancers have been incorporated in the One-step NZYSpeedy RTqPCR Green kit, ROX, including buffer chemistry and a polymerase with hot-start-like activity. This optimized mixture together with the NZYRT mix results in highly sensitive and successful results.