Superscript One-Step RT-PCR with Platinum
Taq from Invitrogen is designed for convenient, one-tube RT-PCR. The kit consists of two main components. The first is an enzyme mix that contains proven enzymes for both first strand cDNA synthesis, Superscript II H-reverse transcriptase, and subsequent PCR amplification steps, Platinum
Taq. The second component is a 2´ buffer that is optimized for both the reverse transcription and PCR amplification reactions. As such, both separate steps of normal RT-PCR reactions can be performed consecutively in one tube.
Setting reactions up is easy: 50 ul reactions contain 10 pg – 1 ug of template RNA, the optimized 2´ reaction buffer, primers at 0.2 uM and 1 ul of the RT/Platinum Taq Mix. I have used Qiagen RNA purification products to generate template for RT-PCR although alternatives such as Trizol® Reagent are also recommended. Most modern PCR machines are sufficiently sophisticated such that both the RT and PCR steps can be programmed to occur consecutively. Reactions can thus be set up, put in a PCR machine, allowed to run and finally analyzed by gel electrophoresis. Efficient reverse transcription can be achieved in 15?30 min at 45?55oC. During this time the Platinum® Taq remains inactivated as it is bound to an antibody that inhibits the enzyme’s action. During the initial denaturation step for PCR at 94°C this antibody is denatured and the activity of Taq is regained. As such, PCR is always ‘hot start’ which increases the efficiency of this process. The activity of the reverse transcriptase is also lost during this step.
This one-step RT-PCR product is directed for one process - the generation of gene specific cDNA. If you want to generate cDNA libraries, other products would probably be more appropriate. The use of oligo(dT) or random primers are not recommended as the RT step would only generate low concentrations of random products. Gene specific RT-PCR has many uses though such as cloning and various forms of quantitative RT-PCR. For my cloning applications I have definitely had good success with this product. In the event that the RT-PCR does not work however, this product also contains magnesium sulfate solutions to aid optimization. Finally a similar product is also available that is optimized for the amplification of long targets of up to 12 kb.
Peter Haggie, Ph.D.
Post-Doctoral Fellow
University of California, San Francisco