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05% McCormick green food coloring, 0.01?mM NaPO4, and 5?mM KCl. Injection of O. fasciatus was done in fourth instar nymphs before the appearance of sexually dimorphic characters. Nymphs were anesthetized using CO2 or with a 4-minute exposure to diethyl ether vapor. Using a front-loaded Selleck Thiazovivin pulled-glass capillary needle, approximately 1?��l of 4?��g/��l dsRNA was injected at the base of the right metathoracic coxa. This location facilitated easy delivery into the hemolymph and no defects were observed at the site after ecdysis. Injection of T. castaneum pre-pupal larvae was as described by Angelini et al. (2009). The extent of gene knockdown was determined using quantitative Icotinib solubility dmso realtime RT-PCR (qPCR) amplification of target gene sequences. For validation of RNAi, expression was compared between gene-specific and nonspecific control dsRNA treatments (GFP). Total RNA was isolated using the PureLink RNA Mini Kit (Life Technologies) from the abdominal tissue (A4-A12) of individual young adult O. fasciatus or 10 pooled 3-day-old T. castaneum pupae. These stages were chosen because they allowed for consistent selection. Isolated RNA was stored at ??80?��C. For all O. fasciatus treatments, at least 3 biological replicates were included. Total RNA concentrations were determined by triplicate measures on a nanoscale spectrophotometer and diluted to 100?ng/��l immediately prior to assays. Total RNA was used as template in reverse transcription/ SYBR Green realtime PCR reactions (Quanta BioSciences). For each gene, exact primers were designed using the Primer3 algorithm (Rozen and Skaletsky, 2000), avoiding conserved functional domains and dsRNA regions. Dissociation curves for each reaction were used to verify that only single products were amplified. To produce quantitative template standards, clones were linearized and transcribed in vitro from T7 promoters to produce single-stranded RNA. This RNA was treated with DNase I to remove template DNA and purified by precipitation in ammonium acetate and ethanol. Immediately before qPCR assays, the RNA concentration was determined in triplicate (as described earlier) and the molar YES1 quantity was calculated based on the size of the RNA. Dilution series were then prepared fresh for each plate at concentrations of 103, 105, and 107 RNA molecules to serve as a standard curve ( Pfaffl, 2004). The degree of knockdown in RNAi specimens is given in Table?2 with statistical significance based on significant difference from control GFP dsRNA treatment in Tukey's honest significant difference (HSD) test (p?
