Purpose – a method for synthesis of iRNA which allows both RNA strands to begenerated simultaneously from a PCR fragment which contains a T7 promoter on eachend.Background - The optimum length of a double-strand RNA (dsRNA) for maximuminterference activity is unclear, but some circumstantial evidence suggests that lengths of700 to 800 bp are most active. Carthew’s lab has found that dsRNAs as short as 200 bpand as long as 2000 bp have potent interfering activities. The dsRNA can be made fromcDNA or genomic DNA templates. Data suggests that for RNAi most of the dsRNAshould correspond to exon regions. dsRNAs with two or more exon regions interruptedby introns will work quite well in Drosophila. One can use dsRNAs corresponding tocoding sequence and/or untranslated regions (UTRs). One approach has been to use adsRNA corresponding to the 5' or 3' UTR (either UTR can mediate interference) since adsRNA to one gene can cross-interfere with another gene's activity if the dsRNA issufficiently similar in coding sequence to the second gene.
Method1. Choose primer sequences that will amplify the region you want to act as the dsRNAtemplate. Consider using MIT's Primer3 program at http://www.genome.wi.mit.edu//cgibin/primer/primer3_www.cgi to choose optimal primer sequences for any given region.Complementary sequences should be 20 to 24 nt in length with a 22 nt optimum and60°C optimum Tm.2. The 5' end of each primer can correspond to either the 27 nt “T7 promoter sequence”(TAATACGACTCACTATAGGGAGACCAC) [Carthew] or the 27 nt “T7 RNApolymerase binding site” (GAATTAATACGACTCACTATAGGGAGA) [Clemens].Thus, each primer will be 47 to 51 nt long (27 + [20 to 24]).3. Perform a 50μl PCR reaction with T7-linked primers and suitable template. Clonedplasmids or phage are optimal, but the method will also work on RT-PCR DNA orgenomic DNA. The first 10 cycles should have a 40°C annealing step, followed by 35cycles with a 55°C annealing step.4. Phenol-chloroform extract and ethanol precipitate in NH4OAc. Dissolve in TE bufferand measure concentration spectrophotometrically. Clemens purified PCR productsusing high pure PCR purification kit [Roche Molecular Biochemicals]5. Perform an RNA synthesis reaction in 50μl volume with 1μg of PCR DNA templateusing T7 RNA polymerase. Most labs use in vitro transcription kits Megascript T7 ormMessage mMachine [Ambion]6. Remove DNA template with RNase-free DNAase [Tuschl used RQ1 Dnase –Promega]. Phenol-chloroform extract and precipitate the RNA with NH4OAc andEthanol.7. Dissolve the dsRNA in TE buffer and measure yield by spectrometer. Typical yields ofRNA from 1 μg DNA template are in the 80 to 100μg range.8. Aliquot and store dsRNA as a NaOAc/ethanol precipitate at -80°C until immediatelybefore use. Carthew’s lab states that the RNA becomes double-stranded during thesynthesis reaction. [NOTE - Some labs perform annealing step. Clemens resuspends inH2O and stores at –20*C.]9. Test the dsRNA’s condition by native agarose gel electrophoresis in TBE.Electrophorese 6-10μg of RNA and stain with ethidium bromide. Alternatively, tracelabelRNA with 32P-ATP during synthesis, and visualize electrophoresed products byautoradiography. [NOTE - Only preparations in which the electrophoretic mobility ofmost of the RNA is shifted to that expected for dsRNA (very close to duplex DNAmobility) of the appropriate length should be used. Sometimes you will observe a smearof higher-order RNAs migrating slower than the dsRNA species. Although these mayconstitute over half of the RNA present, Carthew found that the interfering activities ofthese preparations are similar to more homogeneous preparations