Determination of Mitochondrial Gene Expression by Real-Time PCR.

After perfusion from infected animals, the harvested worms were immediately placed in 0.5 mL of TRIzol (Invitrogen) and mashed using a TissueLyser II (Qiagen). Total RNA was isolated and further purified using an RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. Purified RNA was quantified using a NanoDrop ND-1000 spectrophotometer. First-strand cDNA was synthesized using isolated RNA, SuperScript II reverse transcriptase (Invitrogen), and oligo dT as a primer. Mitochondrial CcO subunit I (CcO I) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FN314248.1","term_id":"226468607","term_text":"FN314248.1"}}FN314248.1) and NADH dehydrogenase (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FN317713.1","term_id":"226480691","term_text":"FN317713.1"}}FN317713.1) were analyzed by qRT-PCR using a LightCycler480 real-time PCR system (Roche, Switzerland) and SYBR green qPCR Master Mixes (Roche). Expression levels of S. japonicum β-actin (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF223400.1","term_id":"6979993","term_text":"AF223400.1"}}AF223400.1) were used as endogenous controls within each sample. β-actin primers were as follows: forward, 5′-AGCGTGGTTACAGCTTCACG-3′ and reverse, 5′-AACGCCTCAGGACAACGGAA-3′. CcO I primers were as follows: forward, 5′-TGGGTTCTATTGTGTGTTTGGG-3′ and reverse, 5′-CACGCAACCCACTACTCCCT-3′. NADH dehydrogenase primers were as follows: forward, 5′-TCTGGAAGCCGCACTTGTTG -3′ and reverse 5′-CGAACCGTCAACAGCAAAGGT-3′. Relative expression was calculated using the 2^−ΔΔCT method.