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 and the amount of starting material can vary from 1 pg to 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.