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. More.

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