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.
The product is tested functionally using 10 pg of total HeLa RNA as the template for amplification of a 353-bp segment of β-actin mRNA (40 cycles). A minimum of 25 ng of the RT-PCR product was obtained under these conditions.