Characterizing Olfactory Transcripts in Insect Heads

We decided to study heads, as they contain most of the organs involved in chemosensory function and feeding, as well as most of the olfactory-associated proteins [47 (link),48 (link)]. Female heads were dissected on ice using a sterile scalpel and pooled in a 1.5 mL microcentrifuge tube (N = 5 per pool). Total RNA was extracted using the RNEasy Plant Mini Kit (QIAGEN, Hilden, Germany) and eluted in 50 μL of RNAse-free water. The integrity of the RNA samples was assessed using a 1.1% gel by denaturing formaldehyde agarose gel electrophoresis, and the concentrations were estimated by spectrophotometry at 260 nm (Epoch Microplate Spectrophotometer, Biotek), resulting in the range of 4.26–8.17 ng/μL of total RNA for all samples. DNA traces were removed from the samples by DNase treatment using Turbo DNase (Ambion). Single-stranded cDNAs were synthesized using the SuperScript III Reverse Transcriptase System (Invitrogen). All procedures were conducted following the manufacturer’s instructions.