Synthesis and Characterization of RmAQP2 dsRNA

Synthesis of double stranded RNA (dsRNA) was performed as previously described [13 (link)]. Specific primers were designed based on the cDNA sequence of the RmAQP2 gene described in the R. microplus Gene Index Project [17 (link)] (Table 1). Two fragments of approximately 400 bp were amplified, one located at the 5′ and another one located at the 3′ of the RmAQP2 gene (Additional file 1: Figure S1). PCR products were cloned into pCR™II-TOPO® (Invitrogen), sequenced and used for in vitro transcription. The MEGAscript® Transcription Kit (Ambion) was used for the dsRNA synthesis following the manufacturer’s protocol. The two RmAQP2 dsRNA molecules were checked by electrophoresis on agarose gel, quantified by spectrophotometry and kept at −20 °C until used for tick injection.