10.2.2. Production of dsRNA

Since dsRNAs can cause off-target effects, you need to be careful in designing them. Nevertheless, RNAi efforts using dsRNA constructs have proven effective in honey bees. To avoid targets that might interfere with other honey bee genes, you need to compare your sequence with the honey bee genome during the design process using the Basic Local Alignment Tool (www.ncbi.nlm.nih.gov). Make sure none of the designed dsRNAs has 20-bp segments identical to any known bee sequence. As dsRNAs are processed by the dicer complex into a cocktail of siRNAs 19–21 nt in length, the absence of 20-nt stretches of homology minimizes the possibility of off-target effects.

  1. Use the E-RNAi web application (Horn and Boutros, 2010) for optimal dsRNA design.
    Design of dsRNA sequences has to be stringent in order to avoid/minimize off-target effects.
  2. Set up appropriate negative controls.
    Note: be careful using GFP; this sequence might cause off-target effects in some cases (GenBank ID: U17997, Clontech ; Jarosch and Moritz, 2012).
    Other possible negative controls: e.g., Q-marker (Beye et al., 2002).
  3. Amplify the chosen target fragment by using target specific T7 (TAA TAC GAC TCA CTA TAG GGC GAT) added primer in optimized PCRs using approximately 100-ng genomic DNA obtained by chloroform– phenol extraction (e.g. Maniatis et al., 1982).
  4. Clone the amplified fragments into pGem-T easy vectors (Promega) according to the manufacturer’s instructions. (Cloning eliminates the possibility of a dsRNA mixture due to a polymorphism of the PCR product).
  5. Transform your plasmids into JM109 competent cells (Promega) following the instructions from the manufacturer.
  6. Prepare the plasmids according to Del Sal (1988).
  7. Analyse the identity of the cloned sequence by Sanger sequencing.
  8. Once the right clone has been identified its insert needs to be amplified to serve as a template for dsRNA production by standard PCR using again T7 tailed primers.
    8.1. E.g. use Biotherm DNA Polymerase (Genecraft); chemicals:
    0.2 mM dNTPs,
    0.3 µM of T7-promotor added primer,
    5 U Taq Polymerase,
    in a total reaction volume of 100 µl.
    8.2. PCR protocol:
    5 min DNA denaturation, and Taq activation, at 95 °C,
    40 cycles of:
    95 °C for 30 sec,
    x°C (primer specific annealing temperature) 30 sec,
    72 °C for 1 min.
    A final extension of 20 min at 72°C completes the protocol.
  9. Purify the PCR-products with the QIAquick® PCR Purification Kit (Qiagen).
  10. Use the T7 RibomaxTM Express RNAi System (Promega) for dsRNA production.
    Time course experiments and experiments for optimizing the incubation temperature have to be conducted beforehand (e.g. Jarosch et al., 2011 used an extended transcription time of 5 h at 32 °C).
  11. Purify the dsRNA by a Trizol® (Invitrogen) - chloroform-treatment following the manufacturers´ instructions.
  12. Resolve the pellet in nuclease free water.
  13. Assess the dsRNA quality and quantity photometrically and by agarose gels or capillary gel electrophoresis.
    The photometric measurement of the OD260/OD280 ratio should be between 1.8 and 2. A lower ratio indicates contamination with proteins. As a contamination with DNA or dsRNA degradation cannot be detected by photometry, visualization of the dsRNA product is necessary. For this 1.5 % agarose gels can be used, see section 3.2.1). A single distinct band should be visible.
  14. Adjust dsRNA concentrations to 5 µg/µl by diluting with insect ringer (54 mM NaCl; 24 mM KCl; 7 mM CaCl2 x 2H2O) right before the injection.