Computational protocol: Reduction in Musca domestica fecundity by dsRNA-mediated gene knockdown

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Protocol publication

[…] Double stranded RNA targeting M. domestica actin-5C (dsActin-5C), ribosomal protein L26 (dsRPL26) and ribosomal protein S6 (dsRPS6) were produced as previously described for the mosquito Aedes aegypti []. Template selection and design of efficient dsRNA primers utilized the eRNAi web service [,] with standard parameters except for selection of “other organism” (Musca data not available for query in the eRNAi tool), amplicon size range 150–250 bp, and primer output to include appended T7 promoter sequences. Accession numbers, primer sequences, and amplicon information are listed in . An Aedes aegypti specific dsRNA (dsAaRPS6) and a dsGFP control was produced by Monsanto Company (St. Louis, MO) and have been described previously []. Primers for M. domestica were synthesized by IDT DNA (Coralville, IA). T7-appended primers were used to amplify the selected regions of actin-5C, RPL26 and RPS6 from M. domestica cDNA produced from females as described below using Platinum™ Taq (Thermo Fisher Scientific, Waltham, MA) and the manufacturer recommended amplification protocol of 94°C for 3 min, 5 cycles of 94°C for 30 sec, 61°C for 30 sec, 72°C for 30 sec, followed by 30 cycles of 94°C for 30 sec, 64°C for 30 sec, 72°C for 30 sec and a final extension at 72°C for 10 min. The appropriate amplicons were gel purified, verified by sequencing (MacrogenUSA, Rockville, MD) and then used for a second round of amplification to produce T7 template. Template was concentrated and washed 2X with 400 μl of nuclease free water (NFW) in a spin concentrator (Amicon 10K MWCO, Millipore, Billerica, MA). Purified template was reverse transcribed to dsRNA using the MegaScript® kit (Ambion, Thermo Fisher Scientific, Waltham, MA). The manufacturer’s protocol was followed with the following exceptions. The entire kit was used (20 reactions) in one tube with 20 μg of template, reverse transcription was allowed to proceed for 24 hours at 37°C, and after purification the resulting dsRNA was concentrated in a spin concentrator to >12 μg/uL as assessed by NanoDrop™ 2000 (Thermo Fisher Scientific, Waltham, MA). Products were maintained at -20°C until dilution for injection. [...] Oviposition assay data was converted to mean clutch size/fly by dividing the total reproductive output (total number of eggs) by number of flies introduced into the laying chamber. For the species specificity experiments, where complete egg clutches were not collected, percentages for each treatment group were calculated relative to dsGFP. Statistical analysis was performed in SigmaPlot v13 (SyStat Software, San Jose, CA). The null hypothesis is that the treatments were not different. Normality was determined by the Shapiro-Wilk test, and statistical significance by ANOVA and means separation was performed using the Holm-Sidak test. A comparison of mortality occurring during the oviposition assay was performed using a Kruskal-Wallis non-parametric analysis due to the non-normal behavior of the dataset.Quantitative PCR data were normalized to single (actin-5) or dual reference genes (actin-5 and L24) in accordance with MIQE guidelines. When using two reference genes, a geometric mean Ct was used as the reference value for calculation of ΔΔCt according to standard methods []. For expression experiments on whole flies at 3 d PI, a sample from the dsGFP injection group was used as the reference sample for each experiment and was included on each plate to allow plate to plate comparison. Comparisons of RE were analyzed in SigmaPlot by t-test when the data were normally distributed and with the Mann-Whitney rank sum test when data were non-normal. […]

Pipeline specifications

Software tools E-RNAi, SigmaPlot
Applications Miscellaneous, Non-coding RNA analysis
Organisms Drosophila melanogaster, Musca domestica, Monodelphis domestica, Aedes aegypti