In Vitro Transcribed (IVT) RNAi

When available predesigned siRNAs do not meet your specific research needs, or you want to do it yourself, in vitro transcription (IVT) of RNAi reagents is an efficient, flexible option. This method allows you to generate custom double-stranded siRNAs in the lab—excellent for targeting novel genes or working in non-traditional model systems.

The MEGAScript RNAi and Silencer siRNA Construction Kits offer a comprehensive solution for synthesizing high-quality RNA reagents, enabling accurate and efficient knockdown of target genes in various experimental settings. Whether you are studying gene function, validating gene targets, or conducting functional genomics research, these kits help provide the reliability and performance required for successful RNA interference (RNAi) experiments. Explore this page to find detailed product information, protocols, and recommendations to optimize your RNAi design and workflow.

The MEGAScript RNAi kit is used to:

• Synthesize dsRNA larger than 200 bp for your RNAi experiments
• Produce up to 20 dsRNAs
• Generate 10–50 times the amount of RNA produced by conventional transcription reactions

The Silencer siRNA Construction Kit is used to:

• Synthesize siRNAs (~20 bp) when predesigned siRNA is not available
• Generate and purify up to 15 siRNAs in less than 24 hours
• Execute hundreds of transfections per reaction

The in vitro transcription method included with the MEGAScript RNAi and Silencer siRNA Construction Kits uses T7 RNA polymerase to generate individual strands of the siRNA.

1. Templates for the reactions are produced from two inexpensive DNA oligonucleotides encoding the desired siRNA strands. These oligonucleotides are designed to include an 8 base sequence complementary to the 3' end of the T7 promoter primer included in the kit.
2. 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.
3. After transcription, the reactions are combined to permit annealing of the two siRNA strands.
4. 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.