The SuperScript™ III First-Strand Synthesis System for RT-PCR is optimized to synthesize first-strand cDNA from purified poly(A)⁺ or total RNA. RNA targets from 100 bp to >12 kb can be detected with this system. The amount of starting material can vary from 1 pg–5 µg of total RNA. SuperScript™ III Reverse Transcriptase is a version of M-MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability. The enzyme is used to synthesize cDNA at a temperature range of 42–55°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 may be used to synthesize first-strand cDNA from a total RNA preparation.

Using the SuperScript™ III First-Strand System
cDNA synthesis is performed in the first step using either total RNA or poly(A)⁺-selected RNA primed with oligo(dT), random primers, or a gene-specific primer. In the second step, PCR is performed in a separate tube using primers specific for the gene of interest. For the PCR reaction, we recommend one of the following DNA polymerases: Platinum™ Taq DNA Polymerase provides automatic hot-start conditions for increased specificity up to 4 kb, Platinum™ Taq DNA Polymerase High Fidelity provides increased yield and high fidelity for targets up to 15 kb, and Platinum™ Pfx DNA Polymerase provides maximum fidelity for targets up to 12 kb.