Quantifying Developmental Transcription in Parasitic Worms

Total RNA was extracted from C. sinensis metacercariae and adult worms using TRIzol reagent (Ambion, Carlsbad, CA, USA) according to the manufacturer’s instructions. The first-strand complementary DNA (cDNA) was synthesized with oligo-d (T) primer using a Power cDNA Synthesis Kit (iNtRON Biotechnology, Gyeonggi-do, Korea) according to the manufacturer’s protocol. Quantitative real-time (qRT)-PCR was performed to detect the developmental transcriptional level of Cslegumain in metacercariae and adult worms. The Oligo 6 program was applied to design the primers, and the gene expressing phosphoglycerate kinase (PGK) was employed as a reference gene [14 (link)] (listed in Table 1). The reaction was performed using the LightCycler 1.5 (Roche, Mannheim, Germany) as follows: the reaction mixture was first heated to 95 °C for 15 min, followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 30 s. The relative transcriptional level was calculated using the 2^−△△Ct method.Table 1

Primers sequences used to amplify the cDNAs by qRT-PCR

Primer	Sequence (5′ → 3′)	Size (base pairs)
Cslegumain F	CTTGCCTTCTCATTGCGTTCT	155
Cslegumain R	ATCTGCTTGGTGTCGGTAGTT
Phosphoglycerate kinase F	GCGGGTGCTTA TGCGAGTTGA	190
Phosphoglycerate kinase R	CACCGGGTTGAGGGAA TA TCT
E-cadherin F	GCTCTTCCAGGAACCTCTGTGATG	82
E-cadherin R	AAGCGATGGCGGCATTGTAGG
N-cadheirn F	AAGGTGGATGAAGATGGCATGGTG	171
N-cadheirn R	TGCTGACTCCTTCACTGACTCCTC
α-actinin 4 F	CCACCATTGCCCGCACCATC	133
α-actinin 4 R	ATGCTGCCTGTCTGCTTCTTGTC
β-catenin F	GGCTCTTGTGCGTACTGTCCTTC	99
β-catenin R	GCTTCTTGGTGTCGGCTGGTC
iNOS F	CAGGGTGGAAGCGGTAACAAAGG	86
iNOS R	CCTGCTTGGTGGCGAAGATGAG
PI3K F	GCACGCCAAGGAATGCTACTAGG	168
PI3K R	GGACAGTAAGAACAGCCACCAACC
AKT F	GCAGGATGTGGACCAACGTGAG	110
AKT R	GCAGGCAGCGGATGATGAAGG