Real-Time PCR has become an increasingly popular technique for analysis of gene expression. There are two primary methods of real-time PCR that can be performed. The first involves including the reverse transcriptase step in the same tube as the PCR reaction (one-step).
The second method involves creating cDNA first by means of a separate reverse transcription reaction and then adding the cDNA to the PCR reaction (two-step). There are advantages and disadvantages to both systems that you should considered before choosing the best one for your application, these include the ease of use and cost of reaction to the resulting yield and sequence representation.