Reverse transcription is a key reaction in the workflow of biomolecular studies, since it enables researchers to effectively know the RNA expression pattern of their biological targets. The ImProm-II™ Reverse Transcription System from Promega offers a reliable way to achieve optimal results in cDNA synthesis. The kit contains everything necessary for cDNA synthesis: reaction buffer, MgCl2, dNTP mixture, ImProm-II™ reverse transcriptase, nuclease-free water, recombinant ribonuclease inhibitor as well as Oligo(dT)15 primer and random primers (other suppliers often sell them separately). Moreover, the kit contains a 1.2 kb kanamycin positive control RNA (and its specific primers) to test the efficiency of the system. Hence, this product contains all the researcher needs to synthesize cDNAs for his PCR-based downstream application.
Reverse transcriptase requires a small amount of starting RNA and so reverse transcription reactions from up to 1ug of total RNA, poly(A)+ mRNA or synthetic transcript RNA can be easily carried out in 20 ul reactions. As it is for other reverse transcription kits, the protocol is divided into two steps: 1) combination of target RNA and primer and denaturation at 70°C for 5 minutes, 2) set up of the reverse transcription reaction for experimental samples and negative control (and positive reaction control, if needed). Once prepared, an aliquot of the RNA and primer mix is added to the transcription reaction (usually 5+15ul, respectively) and the annealing step is carried out. Afterwards, the incubation at 42°C for 60 minutes is performed. Finally, the reverse transcriptase is inactivated at 70°C for 15 minutes.
The kit includes everything needed to perform complete cDNA synthesis. Also, the troubleshooting guide is exhaustive. This is a good kit for researchers who need a reliable system for RNA studies, although in some cases, optimization is required (for example, the MgCl2 concentration must be optimized between 1.5–8.0mM). With brain extract I was using, I usually added MgCl2 to achieve a final concentration between 2.0 and 3.0 mM).
ImProm-II™ Reverse Transcription System from Promega
Efficient system for cDNA synthesis. A large and complete series of useful components. Good troubleshooting guide.
Optimization of MgCl2 is often required.
The Bottom Line
Everything you need to synthesize cDNAs.