Gotaq qpcr master mix reagent system

RNA was isolated using ReliaPrep™ RNA Cell Miniprep System (Promega) following the instructions of the manufacturer. cDNA was synthesized using GoScript™ Reverse Transcription System (Promega) according to the manufacturer's instructions. qPCR was performed in triplicate reactions using GoTaq® qPCR Master Mix reagent system (Promega). The reaction was run on the Applied Biosystems® QuantStudio™ 7 Flex Real‐Time PCR System (Life Technologies). The data were analysed using the QuantStudio™ software (Life Technologies) and relative gene expression was determined using the 2−ΔΔCt method using GAPDH as a housekeeping gene. The list of oligonucleotides is shown in Table S1.

Total RNA was extracted using TRIzol Reagent (Invitrogen, Thermo Fisher Scientific, USA) from whole sexual animals 1 week after the final dsRNA feeding. Total RNA was DNase-treated and purified, followed by cDNA synthesis using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., USA). klf4l primers were designed to sequences outside of the sequence targeted by the dsRNA in order to avoid spurious amplification of dsRNA along with cDNA. For example, primers to the amino terminus of klf4l were used to quantify klf4l knockdown levels in klf4l RNAi (carboxyl terminus) animals in which dsRNA targeting the carboxyl terminus was used, and vice versa. All quantitative PCR (qPCR) analyses were normalized to endogenous control gene β-tubulin. qPCR primers are listed in S2 Table. qPCR was performed using the GoTaq qPCR Master Mix reagent system (Promega, USA) on a StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, USA). Three technical replicates were run for each of 3 biological replicates. ΔΔCt calculations were performed in Microsoft Excel and 2^−ΔΔCt values (normalized to control RNAi) with 95% confidence intervals were plotted using GraphPad Prism software.

Quantitative PCR was performed in triplicate reactions using GoTaq® qPCR Master Mix reagent system (Promega). The reaction was run on the Applied Biosystems® QuantStudio™ 7 Flex Real-Time PCR System (Thermo Fisher Scientific). The data was analysed using the QuantStudio™ software (Thermo Fisher Scientific) and relative gene expression was determined using the 2^−∆∆Ct method using GAPDH as a housekeeping gene. List of primers can be found in Supplementary Table S5.