Quantifying P-glycoprotein Expression in Worms

Expression levels of P-gp genes was determined using qPCR in adult male and female worms. qPCR primers were designed to amplify the genes (Table S1) and specificity was confirmed using BLAST in silico. RNA extracted from pools of adult worms and cell types was extracted with Trizol reagent followed by purification using the Direct-zol RNA Miniprep kit (Zymo Research) according to the manufacturer's instructions. cDNA was synthesized from 50 ng of total RNA in a 20 µL volume using the iScript cDNA synthesis kit (Bio-rad), using random oligonucleotides. qPCR reactions were individually optimized using diluted cDNA synthesized from adult T. canis worms. Specificity was determined using melt curve analysis and sequencing. 18 s RNA was used as a reference gene and amplified using previously described primers^{34 (link)}. qPCR was carried out in technical duplicates in a volume of 20 µL with 2 µL of diluted cDNA, 1 × of SSoAdvanced Universal SYBR Green Master Mix (Bio-rad) and 0.2–0.5 µM of diluted primers. PCR efficiency was determined for each primer pair using LinRegPCR^{35 (link)} and change in gene expression was calculated using the efficiency corrected ΔCt method based on single samples³⁶ .