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).
- 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.
- Immobilise bees by cooling down in at 4°C.
- Fix the bees on wax plates using small fixing pins.
- 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).
- Inject negative controls with insect ringer (54 mM NaCl; 24 mM KCl; 7 mM CaCl2 x 2H2O).
- 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)).
- Sacrifice the bees by shock-freezing in liquid nitrogen.
- Store them at −80°C until tissue preparation.
- Prepare the worker tissues on cooled wax plates using an RNA Stabilization Reagent (e.g. RNAlater®) in order to avoid RNA degradation.