Takara's Two-Step RT-PCR kit, RNA PCR Kit (AMV) Version 3.0, is designed to perform single tube reverse transcription reactions using the AMV Reverse Transcriptase for cDNA synthesis. Subsequent cDNA amplification takes advantage of the properties of Takara Ex Taq HS, a high fidelity, hot-start DNA polymerase.
The Takara RNA PCR kit uses AMV RTase XL, which possesses higher thermostability and a broader range of reaction temperatures (42–60°C) than MMLV RTase. This allows transcription to occur at higher temperatures and helps eliminate potential RNA secondary structures which can impede downstream cDNA amplification. The supplied Oligo dT-Adaptor Primer is designed for highly efficient cDNA synthesis from the poly(A)+ RNA 3'-terminus, allowing later amplification of unknown 3'-termini using 3'-RACE. Components for 3'-RACE are supplied in this two-step RT-PCR kit.
Reverse transcription of RNA expressed at a low level (rare RNAs) may also be performed by creating a 20 µL reverse transcription reaction mixture consisting of up to 9.5 µL RNA sample volume. Takara Ex Taq HS is the enzyme of choice for cDNA amplification because the Takara Ex Taq HS enzyme prevents non-specific room temperature DNA amplification as a result of mispriming and primer dimerization. Amplification of cDNAs up to 5 kb in length is possible using this RNA PCR kit.