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Do I need 1-step or 2-step reaction?


Both the cDNA synthesis and PCR reactions occur in the same mix. This option is suited for high throughput applications due to its speed and ease of set up. There is also a reduced risk of contamination. It is not ideal for low quality RNA samples or if the cDNA is required for archive or separate analysis.


The cDNA synthesis and PCR reactions occur separately. This option is better suited if the cDNA product needs to be retained for analysis. It also allows for higher levels of reaction optimization. It permits control over the type and concentration of enzyme, RNA input and concentration of cDNA which, in turn, results in higher sensitivity compared to the 1-step format.

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