Quantitative Analysis of Schistosoma mansoni Transcripts

Seven days after dsRNA exposure, 1,000 schistosomula were separated for RNA extraction and relative expression analysis by quantitative real-time PCR (RT-qPCR). Schistosoma mansoni cytochrome C oxidase I gene (SmcoxI—Smp_900000) was used as the internal control gene. RNA extractions were performed using the TRIzol Reagent method followed by purification with the RNeasy Mini Kit (Qiagen), according to the manufacturer’s guidelines. RNA samples were treated with the TURBO DNA-free kit (Ambion) to remove residual genomic DNA, quantified using the Nanodrop Spectrometer ND-1000, and stored at −70°C.
For adults, for 7 days, two worm pairs per day were removed and macerated with TRIzol Reagent for RNA extraction as described previously. Experiments were performed in four biological replicates.
The cDNAs were synthesized with equal amounts of the extracted RNAs using the SuperScript II Reverse Transcriptase (Invitrogen), with oligo(dT)18 following the manufacturer’s protocol. Primers for qPCR analysis were designed using the Primer 3 program.³ Primer efficiencies were estimated by titration analysis to be 100 ± 5% (data not shown), and the specificity was verified by the melting curve. qPCR reactions were performed on 7500 Real-Time PCR System (Applied Biosystems) with SYBR Green PCR Master Mix (Applied Biosystems) and 200 nM of each primer in a final volume of 25 μl. Internal controls to evaluate genomic DNA contaminations (RNA samples) and reagent purity (no cDNA) were included in all analyses. The 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)) was used for relative quantification and normalized with SmcoxI. Transcript levels were expressed as a percentage of difference relative to the unspecific (GFP) or negative control.