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RNAi in Non-Mammalian Cultured Cells

Large-scale Transcription of Long dsRNA for RNAi


Large mass amounts of ready-to-use dsRNA for RNAi experiments in non-mammalian systems can now be easily prepared using the MEGAscript RNAi Kit. The kit is based on Ambion's patented high-yield transcription technology and includes reagents for transcription, nuclease digestion and dsRNA clean up. The kit incorporates a single step, high-yield transcription/annealing reaction, followed by removal of DNA, ssRNA, proteins, free nucleotides and buffer. Each kit can generate 1-2 mg of ready-to-use dsRNA.


Large Scale Synthesis and Purification of dsRNA
Long double-stranded RNA (dsRNA) has been used extensively to induce RNA interference (RNAi) in C. elegans and Drosophila melanogaster (see sidebar, "siRNA or dsRNA"). The ability to generate large amounts of clean dsRNA is essential for the success of RNAi experiments in these organisms. The MEGAscript RNAi Kit, based on Ambion's patented MEGAscript large scale transcription technology, allows RNAi researchers to synthesize 50-100 g amounts of dsRNAs of various sizes (see "RNAi: Size Does Matter") in a single reaction. PCR or plasmid DNA templates containing a T7 RNA polymerase promoter can be used to generate RNA transcripts. DNA templates containing T7 RNA polymerase promoters on both ends of the inserted sequence can also be used (Figure 1). These latter templates eliminate the need for a separate annealing step the simultaneous transcription from both
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