Quantitative PCR Analysis of nrps in RNAi Planarians

Quantitative PCR (qPCR) was performed as previously described (17 (link)). Briefly, total RNA was extracted with TRIzol reagent from RNAi planarians that had been starved 1 week after the final dsRNA feeding. Total RNA was DNase-treated, purified (RNA Clean & Concentrator Kit, Zymo Research Corporation), and reverse transcribed into cDNA (iScript cDNA Synthesis Kit, Bio-Rad Laboratories, Inc.). nrps primers (Table S2) were designed to sequences outside of the dsRNA target sequence (i.e., the sequence cloned into pJC53.2). qPCR reactions were set up with GoTaq qPCR Master Mix reagent system (Promega) and run on a StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). Gene expression levels were normalized to the endogenous control gene β-tubulin. The ΔΔCt method was used to analyze relative changes in nrps expression in RNAi knockdowns (4 biological replicates, 3 technical replicates each). 2^−ΔΔCt values (normalized to control RNAi) with 95% confidence intervals were calculated in Microsoft Excel and plotted using GraphPad Prism software.