Kapa sybr fast

Total RNA was isolated from 20 midguts/sample using TRIzol reagent (Invitrogen). cDNA synthesis was performed using High Capacity cDNA Reverse Transcription Kit (Applied Biosciences, Life Technologies, Carlsbad, CA, USA) with random primers and 1 μg of total RNA. qRT-PCR was performed on a Rotor-Gene Q (Qiagen, Valencia, CA, USA) with Rotor-Gene software (version 2.1.0.9) using KAPA SYBR® FAST according to the manufacturer’s instructions. Reactions were performed using three independent biological samples in triplicate and transcript levels were normalized using rp49 as the reference gene. Data were analyzed using the Q-Gene software with Standard Curves, and samples on the same graph were run simultaneously as described in ref. ^{16 (link)}. Primers used are as follows:
Tor F 5′-CGGTTATCCCGCTCAGTACC; R 5′-GGTGATCATAGTCTGGCGCA
rp49 F 5′-CCAGTCGGATCGATATGCTAA; R 5′-ACGTTGTGCACCAGGAACTT

Total RNA was isolated from cells, whole flies or midguts dissected from appropriately staged animals using TRIzol® reagent (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. cDNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosciences) with 1 µg of RNA, random primers and RNaseOUT™ Recombinant Ribonuclease Inhibitor (Thermo Fisher) according to the manufacturer’s instructions. qRT-PCR was performed using KAPA SYBR® FAST according to manufacturer’s instructions on a Rotor-Gene Q (Qiagen, Valencia, CA, USA) controlled with Rotor-Gene software. Reactions were performed in triplicate and normalised gene expression was calculated relative to that of control gene (rp49, also known as rpL32, for Drosophila and β-actin for human) using standard curves in Q-Gene software. Primers used for qRT-PCR from IDT, except detour and dor from GeneWorks (Supplementary Table 2).