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Extracting RNA and cDNA synthesis. Parasite total RNA was extracted by using the Trizol method [22] and treated with RQ1 RNase-free DNase (Promega, Wisconsin, USA). One microgram of extracted RNA was used for RT-PCR, using the SuperScript III enzyme (Invitrogen, California, USA) in 20-μl reactions, according to manufacturer’s recommendations. cDNA was thus synthesised for 60 min at 50 °C, and PCR amplification was then done with the Platinum Pfx CHIR-99021 enzyme (Invitrogen, California, USA) for 35 cycles at the following temperatures: 94 °C for 15 s, 58 °C for 30 s, 68 °C for 3 min, and a final extension step at 68 °C for 5 min. Additional PCR was carried out using non-reverse transcribed RNA as template (negative control) for discarding genomic DNA contamination.
Peptide synthesis. Three 20-amino-acid-long peptides were synthesised, based on portions from the P. vivax RhopH3 deduced sequence (Sal-I). The amino acid sequences, shown in single letter code, were: 186KIYLSSVGTPTSALKNLYLN205, 299RDDVHLVKPQSVWGIPLFTT318, and 792SAGVGTVSTHSPATAARMGL811. A glycine and cysteine were inserted at the N- and C-termini of each peptide to allow polymerisation. The peptides were synthesised using standard solid phase t-Boc/Bzl peptide synthesis strategy [23]. The peptides were lyophilised and then characterised by RP-HPLC and MALDI-TOF MS.