10.3.3. Gene knock-down by abdominal injection of target-specific dsRNA/siRNA

 RNA interfering molecules injected by intra-abdominal injection do not reach every tissue (Jarosch and Moritz, 2011). But especially the fat body can be easily reached by this user friendly method (Amdam et al

., 2003; Jarosch and Moritz, 2011).

  1. Take age-defined workers (see the BEEBOOK paper on miscellaneous methods (Human et al., 2013)).
    Note: newly emerged workers are a little bit more difficult to inject as their abdomen is quite flexible.
  2. Immobilise bees by cooling down in at 4°C.
  3. Fix the bees on wax plates using small fixing pins.
  4. Inject 5 µg of freshly diluted dsRNA or alternatively 3 µg of freshly diluted siRNA (treatment and control dsRNA/siRNA) between the 5th and 6th abdominal segment using a 10 µl microsyringe (e.g. Hamilton).
  5.  Inject negative controls with insect ringer (54 mM NaCl; 24 mM KCl; 7 mM CaCl2 x 2H2O).
  6. Keep the injected workers on wax plates until they recover and keep bees not showing haemolymph leakage (visible on their substrate or as a droplet on the cuticle) together with about 25 nurse bees (1-10 days) in cages (see the BEEBOOK paper on maintaining adult honey bees in vitro under laboratory conditions (Williams et al., 2013)).
  7. Sacrifice the bees by shock-freezing in liquid nitrogen.
  8. Store them at −80°C until tissue preparation.
  9. Prepare the worker tissues on cooled wax plates using an RNA Stabilization Reagent (e.g. RNAlater®) in order to avoid RNA degradation.