( mRNA and a control primer set toveri...)prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification

Conclusions

With Stratagenes ProSTAR RT-PCR systems, accurate cDNA amplification, high sensitivity, high product yield, and long-target capability are attainable. Use one of the two convenient kit formats, the Ultra HF RT-PCR system or the HF One-Tube RT-PCR system, to fulfill a variety of RNA amplification applications, including cDNA cloning and RNA detection/analysis.

Methods

RT-PCR reactions were carried out using the recommended protocols for the ProSTAR Ultra HF and HF Single-Tube RT-PCR systems. For the ProSTAR HF Single-Tube RT-PCR system, 50-l RT-PCR reaction mixtures contained 1X HF RT-PCR buffer, 200 M each dNTP, forward and reverse gene-specific primers (2 ng/l of each), RNA (200 ng of total RNA or 1 ng of bacteriophage MS2 RNA), 1.25 U of MMLV-RT, and 2.5 U of TaqPlus Precision DNA polymerase. Amplifications were carried out in Stratagenes RoboCycler Gradient 96 temperature cycler fitted with a Hot Top assembly, using 200-l thin-walled PCR tubes. The temperature cycling parameters for all targets incorporated the following: For cDNA synthesis, one cycle at 37C for 15 minutes (targets <2 kb) or 30 minutes (targets >2 kb); for PCR, one cycle at 95C for 1 minute; 40 cycles of 95C for 1 minute, 60C for 1 minute, and 68C for 2 minutes (targets <2 kb) or 2 minutes/kb (targets >2 kb); and one final extension cycle at 68C for 10 minutes.

For the ProSTAR Ultra HF RT-PCR system, 10-l cDNA synthesis reactions contained 1X MMLV-RT buffer, 1 mM each dNTP, 10 ng/l of oligo(dT), and RNA (200 ng of total RNA or 1 ng of bacteriophage MS2 RNA). The reaction mixtures were incubated at 65C for 5 minutes, then cooled to room temperature to allow the primers to anneal to the RNA. A total of 10 U of MMLV-RT was added, and cDNA synthesis was carried out at 37C for 15 minutes (targets <2 kb) or 3
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