Figure 5. Real-time quantitative PCR analysis confirmed cercariae gender-associated DNA microarray results.

PCR was performed using a MiniOpticon Real-Time PCR Detection System (Bio-Rad, UK) and SYBR Green I chemistry according to manufacturer's instructions. Briefly, real-time PCR parameters included 40 cycles, fluorescent reading after each cycle and melt curve analysis of individual products at the end of the 40 cycles. Unique oligonucleotide identification number is shown along with BLASTx annotation and gender association. Significant similarity is defined as (E) value≤10⁻⁰⁵. Fold-difference was calculated as described in Methods . Primer characteristics (Tm, annealing temperature and sequence) along with amplicon size are included in the Dataset S1 (RT-PCR Primers). Underlined unique IDs represent gender-associated transcripts identified by the indirect hybridization strategy (parasite cDNA versus parasite gDNA). The italicized unique ID represents a gender-associated transcript identified by the direct hybridization strategy (female cDNA versus male cDNA). Plain text unique IDs represent gender-associated transcripts identified by both hybridization strategies.