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
To ensure that any effects seen in an RNAi experiment are due to the dsRNA itself, it is imperative that the RNA be purified from any contaminants carried over from the transcription reaction. Unlike less complete competing products, the MEGAscript RNAi Kit provides all the reagents necessary to eliminate DNA template, single-stranded RNA (ssRNA), proteins and free nucleotides. After the dsRNA is synthesized, it is digested with DNase I and ssRNA specific RNase (included) to degrade the DNA template and any residual ssRNA, respectively. The dsRNA is then purified away from proteins, free nucleotides, and nucleic acid degradation products using a quick glass fiber filter-based procedure. The kit also comes with a comprehensive Instruction Manual and a control template to assess transcriptional efficiency. Up to 2 mg of dsRNA can be produced from a single kit.
Analysis of the RNAi Effect
Figure 2. dsRNA-Mediated Silencing of Drosophila U2AF50 and hrp48 Gene Expression. (A) Specific reduction of protein expression levels. The RNAi effect was analyzed by Western blot with anti-U2AF50 (50 kDa) and anti-hrp48 (48 kDa; migrates as a doublet) antibodies. An antibody directed against Pab 1 (lower panel) was used as an additional negative control. (B) Specific reduction of mRNA expression levels. Total RNA samples were purified and analyzed using by Northern blotting. Radiolabeled RNA probes were produced from two 300 bp long PCR templates (U2AF50 and hrp48) or from Ambion pTRI RNA 28S plasmid. The latter probe was used to normalize the amount of RNA added. (C) Dose sensitive reduction of hrp48 protein expression.
Visualizing Introduced dsRNA in Cells
Figure 3. Fluorescently Labeled dsRNA Retains Functionality. (A) Silencing experiments and Western blot analyses were performed as described in Figure 2 three days after treatment with 10 nM of the indicated labeled or unlabeled dsRNA. (B) dsRNA specific for U2AF50 was labeled with Cy3 using the Silencer™ siRNA Labeling Kit, introduced into Drosophila L2 cells, and the cells were analyzed by fluorescence microscopy. BLUE: DAPI stained nuclei. RED: Cy3 labeled U2AF50 dsRNA.
The Silencer Cy3 and FAM siRNA Labeling Kits can be used to label siRNA and long dsRNA, and are the perfect complement to the MEGAscript™ RNAi Kit. Labeled dsRNA can be used to analyze subcellular localization, stability, and uptake. In addition, fluorescently labeled dsRNA is particularly well-suited for double label experiments (with a labeled antibody) to track cells that take up dsRNA and to correlate uptake with down-regulation of the target protein. Although the Silencer siRNA Labeling Kits were developed for labeling siRNA, they can be readily adapted for labeling long dsRNA.
1. Brown D, Jarvis R, Pallotta V, Byrom M, and Ford L. (2002) RNA Interference in Mammalian Cell Culture: Design, Execution and Analysis of the siRNA Effect. Ambion TechNotes 9(1):3 5.
2. Jarvis R and Ford L (2001) The siRNA Target Site is an Important Parameter for Inducing RNAi in Human Cells. Ambion TechNotes 8(5):3 5.
3. Byron M, Pallotta V, Brown B, and Ford L. (2002) Visualizing siRNA in Mammalian Cells: Fluorescence Analysis of the RNAi Effect. Ambion TechNotes 9(3):6 8.
Cy™3 is a trademark of Amersham Biosciences.
The Cy3 and Fluorescein Labeling reagents manufactured for Ambion by Mirus Corporation.