Howlett et al. BMC Genomics 2012, 13:211 http://www.biomedcentral.com/1471-2164/13/211 (Felsenstein, 2010). A Neighbor-Joining cluster was gen- erated for each alignment using the JTT model without rate variation across sites. Bootstrapping was performed by generating 1000 resampled alignments with the Seq- Boot program of the Phylip package and clustering each of them in the same manner as the original alignment. The 1000 clusters were then used to generate a majority rule consensus tree. For phylogenetic analysis of gustatory receptors, pre- dicted amino acid sequences were aligned using Clus- talW with default parameters in Mega 5.04. A neighbor-joining cluster was generated, with bootstrap values calculated by resampling 1000 times, and a consensus tree generated, all using default parameters in Mega 5.04. The following sequences were used: HvCr1, CAD31850.1; DmGr64f, NP_728924.2; DmGr5a, BAB68248.1; DmGr43a, NP_001036531.1; HvCr4, CAD31946.1; HvCr5n, AJ487480.1; BmGr9, EU769120.1; BmGr8R, NM_001130872; BmGr7, BK006594. Remaining Bombyx sequences are from [39]. Preparation of dsRNA A 957 bp fragment of the Msex\Orco open reading frame was amplified from genomic DNA using the fol- lowing primers 1893A5a, 5’- TATAACCATGCAATAA- CAAA-3’; 1893A3a 5’-AGTAACCTGGGAAAAATAAT- 3’ and subcloned into the pDrive vector (Qiagen, cat #231122) according to manufacturers instructions. dsRNA was prepared as recommended by the Drosoph- ila RNAi Screening Center (flyrnai.org and [40]. Briefly, the subcloned fragment was PCR amplified to append T7 sites to each end (primers: 5’-TAATACGACTCAC- TATAGGGTATAACCATGCAATAACAAA-3’ and 5’- TAATACGACTCACTATAGGGAGTAACCTGGGAAA AATAAT-3’). Both strands of the resulting product were in vitro transcribed with the Ambion MEGAscript kit using T7 RNA polymerase according to manufacturer’s instructions (Ambion, cat #AM1333). RNA was purified by LiCl/ethanol precipitation and redissolved in depc- dH2O. RNA strands were annealed by incubating in a boiling water bath and immediately allowing to slow- cool to <30°C, followed by dilution in depc-dH2O to 1 μg/μL. dsRNA was used immediately or aliquoted and stored at −80°C until use. dsRNA corresponding to a 208 bp fragment of MsOR-30 was generated following the same procedure. Template DNA was amplified from genomic DNA using the following primers Fwd: 5’- TTCGCAGTTCAAGAAGAGCA-3’ and Rev: 5’-TTC GTGCATATATTTTTGAAAGTGA-3’. dsRNA delivery Individual larvae were collected on the last day of the fourth larval instar, placed in separate containers, and starved overnight. On the first day of the fifth larval instar, Page 8 of 10 dsRNA was delivered by feeding starved caterpillars a small pellet of food that had been injected with 3 μg Msex \Orco dsRNA (1 μg/μL in depc-dH20) or with 3μL depc- dH20. After 5–6 hours, larvae that had consumed the en- tire food pellet were kept for subsequent analysis; they were returned to an unrestricted diet and housed indi- vidually until tissue collection. For paired samples (dsRNA treated and control), larvae were collected either on the same day or within a few days of one another, with the ex- ception of 2 trials, in which samples were collected 3 or 5 weeks apart. For Msex\Orco dsRNA trials, all larvae were males; for MsOR-30 trials, all larvae were females. RNA extraction, cDNA synthesis and qPCR was carried out in parallel for each paired sample. Quantification of gene expression Chemosensory tissues were harvested at 24, 72 or 120 hours after dsRNA delivery for quantification of Msex \Orco expression. Larval antennae (antennae preps) or maxilla (maxilla preps) were dissected and combined from 2–5 individuals of the same sex into a single tube. The sex of each larva was determined as previously described [41]. Dissected tissues were immersed imme- diately in Trizol reagent (Invitrogen, 15596–026) and tis- sue was homogenized with a micro-pestle and stored at −80°C until RNA extraction. Total RNA purification was performed according to manufacturer’s instructions. First strand cDNA synthesis was performed using ran- dom hexamer primers and the AMV First Strand cDNA synthesis kit (Invitrogen, 12328–032) according to man- ufacturer’s instructions. Real-time quantitative RT-PCR was performed using the SYBR Green method with a Lightcycler 480 instrument (Roche Applied Sciences). Primers were designed with Pri- mer3 [42] to amplify both MsOR2 and the 16 S ribosomal protein transcript rpS3 [43], which was used as an en- dogenous control for relative quantification of expression. The primer sequences were as follows: Msex\Orco-Fwd1: 5’-GAACACTTGTCCGAGGGTGT-3’, Msex\Orco-Rev1: 5’-ACTGGGTTGAACGCCATAAG-3’; rpS3-Fwd2: 5’- GCAGAAGCGGTTCAACATC-3’, rpS3-Rev2: 5’- AGA CCTCCAATGAGTTTGTATC-3’; MsOR-30 Fwd: 5’- CA AAGGAACACGAAAGACGA,-3’, MsOR-30 Rev: 5’- CG ACCACAATAACCACCGTA-3’. The specificity of each pair was confirmed by melt-curve analysis and efficiency was calculated over a 50-fold cDNA dilution range (2.050 for Msex\Orco, 1.977 for rpS3 and 1.965 for MsOR-30 primers). Experimental reaction conditions were as fol- lows: 20μL reactions were prepared with SYBR Green master I mix (Roche, 04 707 516 001), 20pmol each pri- mer, and 0.5μL cDNA template, no-RT control template, or dH20 negative control. Reactions were carried out for a varied number of cycles (5 min 95°C incubation followed by cycles of 10 sec at 95°C, 10 sec at 57°C, 16 sec at 72°C