What is the difference between a one-step and a two-step real-time PCR reaction?
In a one-step reaction the reverse transcription reaction and the real-time PCR reaction are done in one tube, making this a closed tube assay, so contamination can be avoided. It saves pipeting steps and time, and is easy in handling, making it ideal for high throughput screening. In a two-step reaction the reverse transcription reaction and the real-time PCR reaction are done in separate tubes. It gives a more flexible way of working in that the cDNA can be used for more than one real-time PCR reaction and can be archived, eliminating the need to continually isolate RNA. For convenience Bioline sells the cDNA Synthesis Kit (BIO-65025) separately if you wish to take a two-step approach.
