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Five Ways to Produce siRNAs

Choose the Best Method for Your Research


Many researchers now use small interfering RNAs (siRNAs) to reduce the expression of specific mammalian genes. Here we describe five methods for producing siRNAs for use in mammalian RNAi experiments. Each of these methods has its advantages and drawbacks. The best method for generating siRNAs will depend on the goals of the experiment. This article briefly describes the five methods, presents their advantages and disadvantages, and discusses the types of applications for which they are best suited.


Currently, there are five methods for generating siRNAs for gene silencing studies:

1. Chemical synthesis

2. In vitro transcription

3. Digestion of long dsRNA by an RNase III family enzyme (e.g. Dicer, RNase III)

4. Expression in cells from an siRNA expression plasmid or viral vector

5. Expression in cells from a PCR-derived siRNA expression cassette

The first three methods involve in vitro preparation of siRNAs that are then introduced directly into mammalian cells by lipofection, electroporation, or other technique. The last two methods rely on the introduction of DNA-based vectors and cassettes that express siRNAs within the cells. Table 1, below, summarizes these methods.

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