Method #3: Digestion of Long dsRNAs to Create an siRNA Cocktail
One of the major drawbacks of all the other methods of siRNA production is the need to design and test several siRNA sequences before an effective one can be identified. Preparation of siRNA cocktails overcomes this limitation. In this method, long dsRNAs are prepared by in vitro transcription using a template that typically encodes a 2001000 nt region of the target mRNA. The dsRNA is then digested in vitro with RNase III (or Dicer) to produce a population, or cocktail, of siRNAs. Since the cocktail contains many different siRNAs, efficient gene knockdown is virtually guaranteed. A representative of one of these experiments is depicted in Figure 2.
The major benefit of this approach is the ability
to bypass the testing steps involved in selecting an effective siRNA sequence,
saving researchers both time and money (note that RNase III reactions
are typically less expensive than those performed with Dicer). One downside
of this approach, however, is the theoretical potential for nonspecific
silencing effects, particularly for closely related genes. Most research
to date indicates that this does not pose a problem (1-4).
Fast and inexpensive analysis of loss of function phenotypes
Not suited for:
Long term studies or studies that require a single, defined siRNA sequence
Ambion's Solution: Silencer siRNA Cocktai