Femtojet express

According to our previous methods [9 (link),31 (link)], rats were intraperitoneally (i.p.) injected with pentobarbital (65 mg/kg) under isoflurane (2%) anesthesia. In brief, we inserted the microcapillary backfilled with rAAV solution into the SDH between Th13 and L1 vertebrae (500 μm lateral from the midline and 250 μm in depth from the surface of the dorsal root entry zone) and microinjected 800 nl rAAV solution (3 × 10¹² GC/mL) using FemtoJet Express (Eppendorf, Hamburg, Germany). After microinjection, we slowly removed the inserted microcapillary from the SDH and the skin was sutured with 3–0 silk.

For parental RNAi in Tribolium, 300-400 female G10011 pupae (at 70-80% pupal development) were injected with dsRNA (2 µg/µl) or injection buffer only (control) using a FemtoJet Express (Eppendorf). Injected pupae were placed on fine wheat flour (type 405) for 24 h at 28°C. Eclosed beetles were added to ∼200 male G10011 beetles and maintained for another 24 h at 28°C. Then, all beetles were collected, placed on fresh fine wheat flour and shifted to 32°C. Eggs were collected for 24 h, aged for an additional 48 h and then fixated for immunostaining. Eggs were collected for eight consecutive days. Pupal injections were performed twice with fragment 1 and once with fragment 2 (see following section).

The templates for the bidirectional in vitro dsRNA synthesis were generated by PCR using a T7 and an SP6 primer with a T7 promoter sequence overhang. The dsRNA was then synthesized by using the T7 Megascript Kit (Ambion, Life Technologies GmbH, Darmstadt, Germany) with the respective PCR fragment as the template. After DNAse treatment and LiCl precipitation, the RNA was annealed by boiling for about 20 min cooling down to room temperature over 3 h. The formation of dsRNA was confirmed by gel electrophoresis and the concentration was adjusted to 3 µg/µL with injection buffer (1.4 mM NaCl, 0.07 mM Na₂HPO₄, 0.03 mM KH₂PO₄, 4 mM KCL, and 10% phenol red), using a NanoDrop ND-100 (NanoDrop Technologies, Inc., Wilmington, NC, USA). The mock dsRNA was made from a PCRII vector containing a 427 bp fragment of DsRed (kindly provided by G. Bucher).
For the injection of dsRNA, late pupae were mounted with double sided adhesive tape on an object slide and approximately 0.5 µL of the dsRNA solution was injected into the conjuctivum between the fourth and fifth abdominal segments using a pulled glass capillary coupled to a FemtoJet express (Eppendorf, Hamburg, Germany). The injected pupae were placed in flour filled Petri dishes and stored in an incubator at 30 °C until adult beetles emerged, which were then aged and food-deprived before their use for EAG experiments.