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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 ß-actin
control (Figure 1B and C). These results suggested that Gene 1
does indeed positively regulate the expression
of Gene 2.
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| 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) ß-actin expression (internal
control). Transfections were carried out as described
in panel A. |
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