The One-Step RT-PCR with HotStar Taq System is designed for the convenient, sensitive, and reproducible detection and analysis of RNA molecules by RT-PCR. Components for both cDNA synthesis and PCR are combined in a single tube, using gene-specific primers and target RNAs from either total RNA or mRNA. Reverse transcription automatically follows PCR cycling without additional steps. The kit consists of two major components: RT/ HotStar Taq Mix and 2X Reaction Mix. The RT/ HotStar Taq Mix contains a mixture of Reverse Transcriptase and HotStar Taq DNA Polymerase for optimal cDNA synthesis and PCR amplification. Reverse transcriptase is a modified version of Moloney Murine Leukemia Virus (M-MLV) RT, engineered to reduce RNase H activity and increase thermal stability. HotStar Taq DNA polymerase is Taq DNA polymerase complexed with a proprietary antibody that inhibits polymerase activity at ambient temperatures. The antibody is denatured and polymerase activity is restored during the denaturation step in PCR cycling at 94°C. This provides an automatic “hot start” in PCR, increasing sensitivity, specificity, and yield. The 2X Reaction Mix consists of a proprietary buffer system optimized for reverse transcription and PCR amplification, Mg2+ optimized for universal use, deoxyribonucleotide triphosphates, and stabilizers. This convenient 2X format allows further addition of template and primer at any desired concentration. One addition tube of MgSO₄ (50 mM) is included in the kit. Reagents are provided with the sufficient amplification reactions of 50 μl each.