Project description:The NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to screen the resulting mRNA harvested from ticks treated with the tick Ubiquitin-63E 600bp dsRNA in comparison to controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A stringent bioinformatics analysis was undertaken to predict off target effects (OTE) resulting from the in silico dicing of the 600bp Ubiquitin-63E dsRNA which identified 10 down (including TC6372) and 13 up-regulated ESTs within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196bp and 109bp dsRNAs, and a cocktail of short hairpin RNAs targeting Ubiquitin-63E demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative real time PCR analysis confirmed TC6372 and predicted off target differential expression. Our study demonstrates the effectiveness and specificity for utilising ~100bp dsRNA products for targeted tick gene knockdown with nil identified off targets.
