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Your Data: Silencing Neural Cell Adhesion Genes with In Vitro Transcribed siRNAs

Ambion has developed a system for siRNA synthesis and purification based on in vitro transcription, the Silencer siRNA Construction Kit (patent pending). This kit produces transfection-ready siRNA at a fraction of the cost of chemical synthesis. Here, researchers at the Salk Institute describe the successful silencing of two neural cell adhesion proteins using siRNAs produced by this kit.

Drs. Qi Chen and David Schubert at the Salk Institute recently used in vitro transcribed siRNAs, prepared with Ambion's Silencer siRNA Construction Kit, to study two genes that are involved in nerve cell adhesion. These researchers sought to determine whether siRNA-mediated silencing of Gene 1 would affect the protein expression levels of a putative downstream gene, Gene 2. To this end, the researchers prepared six siRNAs by in vitro transcription that targeted Gene 1. These siRNAs were transfected individually into the nerve cell line B103, and the protein expression levels of both genes were monitored by Western blot 96 hours post-transfection.

One of the six gene specific siRNAs effectively reduced Gene 1 protein levels (Figure 1A). Interestingly, the same siRNA also reduced the protein levels of Gene 2, but not those of the -actin control (Figure 1B and C). These results suggested that Gene 1 does indeed positive ly regulate the expression of Gene 2.

Figure 1. Gene 1-Specific siRNAs Reduce Expression of Proteins 1 and 2. (A) B103 cells were transfected with Gene 1 specific siRNAs prepared with the Silencer siRNA Construction Kit (6 pmol and 120 pmol). Expression levels of Gene 1 were determined by Western blots 96-hours post-transfection. (B) Gene 2 expression. Transfections were carried out as described in panel A. (C) -actin expression (internal control). Transfections were carried out as described in panel A.

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Cat# Product Name Size 1620 Silencer siRNA Construction Kit 15 siRNA synthesis rxns


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