Adult worms were electroporated with either an siRNA (10 µg) targeting SmAP (SmAP siRNA 1: 5’-AAGAAATCAGCAGATGAGAGATTTAAT-3’), or with a control siRNA that targeted no sequence in the schistosome genome (Control: 5’-CTTCCTCTCTTTCTCTCCCTTGTGA-3’) or with no siRNA, as described previously (22). To assess the level of target gene suppression, RNA was isolated from some worms from each group two days later using the TRIzol Reagent (Thermo Fisher Scientific, MA), as per the manufacturer’s guidelines. Residual DNA was removed by DNase I digestion using a TurboDNA-free kit (Thermo Fisher Scientific, MA). cDNA was synthesized using 1 µg RNA, an oligo (dT)20 primer and Superscript III RT (Invitrogen, CA). Gene expression of SmAP was measured by quantitative real time PCR (RT-qPCR), using custom TaqMan gene expression systems from Applied Biosystems, CA using primers and probes, as previously (23, 27). Alpha tubulin was used as the endogenous control gene for relative quantification, as described (28) employing the ΔΔCt method (29). Results obtained from parasites treated with the irrelevant, control siRNA were used for calibration. For graphical representation, 2-ΔΔCt values were normalized to controls and expressed as percent difference.

To look for any impact of gene suppression on the ability of worms to cleave vitamin B6, PLP was added to living worms in assay buffer seven days post siRNA treatment and free phosphate levels were measured at selected time points following the protocol described above.

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