The analysis of Hirudo medicinalis genome allowed in silico prediction of mRNA databases according to intro-exon boundaries. Based on the candidate sequence detected, forward and reverse primers were designed to frame the complete sequence of predicted mRNA. From total RNA extracted from leech nerve cord using TRIzol® reagent and according to the manufacturer’s procedure (Invitrogen, USA), cDNA were synthesized using an oligo(dT) priming. The calreticulin-related molecule was amplified by PCR using the specific forward (5′GGTAGCAATACGTGCAGTTTG3′) and reverse (5′GCAACCAAGAGTAGGCAACC3′) primers and the Platinum®Taq DNA Polymerase according to the manufacturer’s instructions (Invitrogen, USA). Selected PCR products were ligated into pGEM T-easy vector and cloned into JM109 cells according to the manufacturer’s instructions (Promega, USA). Finally, products were sequenced using BigDye Terminator v3.0 polymerization kit before detection on Genetic Analyzer (Applied Biosystems, USA). BLAST programs were used for sequence analysis in databases and comparison with initial predicted mRNA sequence [27 (link),28 (link)]. Phylogenetic analysis was carried out by Geneious® Basic v5.6 software [29 (link)].