The Smcarm1 coding sequence was obtained from the GeneDB database.2 The T7 promoter sequence was added to the 5′-end of primers designed to amplify a template of 569 bp for double-stranded RNAs (dsRNAs) synthesis. A fragment of 360 bp from the green fluorescent protein (GFP) cloned in the pCRII plasmid vector, was used as non-schistosome RNA interference (RNAi) control. The PCR amplified fragments were purified with the QIAquick Gel Extraction KIT (QIAGEN) and used for dsRNA synthesis using the T7 RiboMAX Express RNAi System kit (Promega) according to the supplier’s protocol; except for the time of reactions which was changed to 16 h. DsRNAs annealing and integrity were verified in 1% agarose gel electrophoresis, and the quantification was estimated in a Nanodrop Spectrometer ND-1000 (Thermo Fischer Scientific).
Approximately 2,000 schistosomula were exposed to 100 nM of dsRNAs (Smcarm1 or GFP), immediately after mechanical transformation, in 24-well plate and incubated for 7 days at 37°C, 5% CO2, and 95% humidity with 2 ml of GMEM supplemented as previously mentioned.
Eight adult worms (males and females, separately) were placed in 100 μl of RPMI 1640 medium with 25 μg of dsRNA. The worms were electroporated with specific Smcarm1-dsRNA or unspecific GFP-dsRNA into 4 mm cuvettes at 125 V for 20 ms and cultivated in 24-well plate with 1 ml RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 2% penicillin/streptomycin. Similarly, to count the number of eggs laid, eight worm pairs were electroporated and cultured in six-well plate and the medium was changed daily.
Approximately 2,000 schistosomula were exposed to 100 nM of dsRNAs (Smcarm1 or GFP), immediately after mechanical transformation, in 24-well plate and incubated for 7 days at 37°C, 5% CO2, and 95% humidity with 2 ml of GMEM supplemented as previously mentioned.
Eight adult worms (males and females, separately) were placed in 100 μl of RPMI 1640 medium with 25 μg of dsRNA. The worms were electroporated with specific Smcarm1-dsRNA or unspecific GFP-dsRNA into 4 mm cuvettes at 125 V for 20 ms and cultivated in 24-well plate with 1 ml RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 2% penicillin/streptomycin. Similarly, to count the number of eggs laid, eight worm pairs were electroporated and cultured in six-well plate and the medium was changed daily.
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