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prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification

lexity; Figure 2) or 10 pg (high complexity; data not shown) of reverse-transcribed total RNA. Even low-abundance transcripts can be detected, as shown by the successful amplification of 1.8- to 7.6-kb fragments of human dystrophin from reverse-transcribed total RNA (200 ng of input RNA) (Figure 1).

Figure 2

ProSTAR HF Single-Tube RT-PCR System

The ProSTAR HF Single-Tube RT-PCR system includes all reagents necessary to carry out cDNA synthesis and high-fidelity PCR amplification in a convenient one-tube format (MMLV-RT, TaqPlus Precision DNA polymerase, HF RT-PCR buffer, dNTPs, and RNase-free water). Simply combine reagents into one tube along with gene-specific primers; cDNA synthesis and PCR amplification take place successively during an uninterrupted thermal-cycling program.

Figure 3

With this system, accurately amplify a broad range of target sizes can be accurately amplified from a variety of RNA sources (Figure 3). The kits TaqPlus Precision DNA polymerase blend permits high product yields for amplifications of both relatively short targets (300 to 550 bps; Figure 3, Panel A) and long targets from low-abundance message (1.8- to 5.9-kb dystrophin fragments; Figure 3, Panel B, Lanes 2 to 4). To demonstrate the systems sensitivity, a 325-bp RT-PCR product was amplified from as few as 10 molecules of an in vitro transcribed RNA (Figure 4). In addition, as little as 50 pg of total RNA was sufficient for amplifying the 1.8-kb dystrophin fragment from total RNA (low-abundance message; data not shown).

Figure 4

Both the ProSTAR systems also include test
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