In Vitro Transcribed siRNA
Small interfering RNAs (siRNAs), consisting of ~21 bp double stranded RNAs with 3' dinucleotide overhangs, are now commonly used to induce gene specific silencing via the RNA interference pathway in mammalian cells. The most effective siRNAs can reduce target gene expression by >90%. Unfortunately, some siRNAs have minimal or no effect. To find an siRNA that can efficiently induce gene knockdown, 3–5 siRNAs per gene are typically designed and tested. Thus studies involving the knockdown of several different genes require the synthesis and testing of many different siRNAs.
Reduce siRNA Preparation Time and Costs
When chemically synthesized siRNAs are used, the costs for a single experiment can quickly add up. In addition, turnaround times for custom siRNAs are often several days. The Silencer™ siRNA Construction Kit produces transfection-ready siRNA at a fraction of the cost of chemical synthesis. The kit is based on a patent-pending in vitro transcription method and can generate up to 15 purified siRNAs in less than 24 hours. The cost and time savings gained using the Silencer siRNA Construction Kit enable screening of more genes and more potential targets within your gene to find the most potent siRNA.
How the Kit Works
The in vitro transcription method included with the Silencer siRNA Construction Kit uses T7 RNA polymerase to generate individual strands of the siRNA. Templates for the reactions are produced from two inexpensive DNA oligonucleotides encoding the desired siRNA strands. These oligonucletides are designed to include an 8 base sequence complementary to the 3' end of the T7 promoter primer included in the kit. The oligonucleotides are each annealed to the T7 promoter primer, and a fill-in reaction with Klenow fragment generates a double-stranded template ready for use in the in vitro transcription reaction. After transcription, the reactions are combined to permit annealing of the two siRNA strands. The siRNA preparation is then treated with DNase (to remove template) and RNase (to polish the ends of the double-stranded RNA), and then column purified. The entire procedure requires very little hands on time and can be completed in less than 24 hours.
A Complete siRNA Synthesis Solution
The Silencer siRNA Construction Kit includes all necessary reagents to generate and purify up to 15 double-stranded siRNAs, ready for transfection. Also included are a detailed Instruction Manual and a GAPDH siRNA control template. The GAPDH-specific siRNA made in the control reaction can be used for optimizing transfection protocols.
For help identifying potential target sequences within your mRNA, use our siRNA Target Finder (at Ambion.com). Resulting target sequences can then be sent directly to our Silencer Construction Kit Template Design Tool for the generation of the oligonucleotide templates needed for use of this kit. Or go straight to the Template Design Tool (at Ambion.com) and paste in your own siRNA target sequences.