SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR provides the high-temperature capability of SuperScript® III Reverse Transcriptase in an optimized SuperMix format for the synthesis of first-strand cDNA for use in real-time quantitative RT-PCR (qRT-PCR). The simple, time-saving reaction set-up uses just two tubes: a 2X Reaction Mix and an Enzyme Mix.
SuperScript® III Reverse Transcriptase, included in the RT Enzyme Mix, is a version of M-MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability. The enzyme can be used to synthesize cDNA at a temperature range of 42–60°C, providing increased specificity, higher yields of cDNA, and more full-length product than other reverse transcriptases. Because SuperScript® III RT is not significantly inhibited by ribosomal and transfer RNA, it can be used to synthesize cDNA from total RNA. RNaseOUT™ Recombinant Ribonuclease Inhibitor, also included in the enzyme mix, is an RNase inhibitor protein that safeguards against the degradation of target RNA due to ribonuclease contamination of the RNA preparation.
The 2X RT Reaction Mix includes oligo(dT)20, random hexamers, MgCl2, and dNTPs in a buffer formulation that has been optimized for qRT-PCR. E. coli RNase H is provided as a separate tube in the kit to remove the RNA template from the cDNA:RNA hybrid molecule after first-strand synthesis. This has been shown to increase sensitivity in qRT-PCR.
Using SuperScript® III First-Strand Synthesis SuperMix
This SuperMix formulation can be used to quantify fewer than 10 copies of a target gene in qRT-PCR, with a broad dynamic range that supports accurate quantification of high-copy mRNA from up to 1 µg of total RNA. Reagents are provided for 50 or 250 RT reactions of 20 µL each.