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Products for this protocol

• Acidic phenol:chloroform (phenol:chloroform:isoamyl alcohol 25:24:1)
   
  
• Ammonium acetate (7.5 M)
   
  
• cDNA fragment of the gene of interest in plasmid with SP6 and T7 promoters
   
  
• Chloroform:isoamyl alcohol (24:1)
   
  
• DEPC-treated H₂O
   
  Add 1ml of fresh DEPC to 1L of H₂O. Shake well to disperse the DEPC through the H₂O. Incubate at 37°C for at least 12 hours and/or autoclave at 15 psi on liquid cycle for 20 minutes to inactivate the remaining DEPC.
   
  
• DNase I (10 units/µl) (Roche)
   
  
• EDTA (0.5 M, pH 8.0)
   
  To prepare EDTA at 0.5 M (pH 8.0): Add 186.1 g of disodium EDTA·2H₂O to 800 ml of H₂O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.
   
  
• Ethanol (absolute and 70%)
   
  
• Phosphate-buffered saline (PBS)
   
  To prepare 1 liter of PBS(Phosphate-buffered Saline), dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na₂PO₄, and 0.24 g of KH₂PO₄ in 800 ml of distilled H₂O. Adjust the pH to 7.4 (or 7.2 if required) with HCl. Add H₂O to 1 liter. Dispense the solution into aliquots and sterilize them by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle or by filter sterilization. Store the buffer at room temperature. If necessary, PBS may be supplemented with 1 mM CaCl₂ and 0.5 mM MgCl₂. Can be made as a 10x stock.
   
  
• RNase I
   
  
• RNAsin (30 units/µl)
   
  
• SP6 polymerase (10-20 units/µl)
   
  
• T7 polymerase (15 units/µl)
   
  
• T7 and SP6 transcription buffer (as provided by the manufacturer)
   
  

• Gel electrophoresis equipment
   
  
• Vortex
   
  
• Water bath
   
  

1. Linearize the cDNA with appropriate restriction endonucleases and then carry out in vitro RNA synthesis from T7 and SP6 promoters as described in Step 2.
    
   
2. For each synthesis reaction (T7 and SP6), assemble the following reagents in a microcentrifuge tube:
    
   
   • Linearized plasmid DNA 2 µg
      
     
   • NTPs 4 mM
      
     
   • T7 polymerase (15 units/µl) or SP6 polymerase (10-20 units/µl) 2 µl
      
     
   • RNAsin (30 units/µl) 0.5 µl
      
     
   • T7 or SP6 transcription buffer 1X
      
     
   • H₂O to 20 µl
      
     
   Incubate for 2 hours at 37ºC. Collect 1 µl of the samples and keep for analysis.
    
   
3. Stop the transcription and add 2 µl of DNase I to remove template DNA. Incubate for a further 30 minutes at 37ºC. Remove 1 µl from each of the reactions and set aside for analysis.
    
   
4. Add 20 µl of DEPC-treated H₂O and mix well. Add a mixture of 2 µl of 0.5 M EDTA and 22 µl of ammonium acetate. Extract the RNA with 1 volume of acidic phenol:chloroform (phenol:chloroform:isoamyl alcohol 25:24:1) followed by extraction with 1 volume of chloroform:isoamyl alcohol (24:1).
    
   
5. Precipitate the RNA with ethanol (by adding 2.5 volumes of absolute ethanol). Wash the pellet with 70% ethanol, then air-dry and dissolve in 20 µl of PBS. For quality control, analyze 1 µl of each sample by gel electrophoresis.
    
   
6. Mix equal amounts of sense and antisense RNA, heat in a water bath to 95ºC for 5 minutes, and reanneal by cooling slowly over several hours. This is most easily achieved by simply turning off the water bath. Monitor the annealing and quality of the dsRNA by electrophoresis under nondenaturing conditions and after digestion with 0.5 µg/ml of RNase I.
    
   
7. Samples can be stored at -70ºC for several weeks.