Fly Intestine RNA Extraction and qRT-PCR

Fly intestines were dissected and transferred to cold phosphate-buffered saline (PBS) buffer. A total of 40 intestines were homogenized with TRIzol reagent (Thermo Fisher, Waltham, MA), and RNA was extracted as previously described [29 (link)]. Ten micrograms of total RNA were reversely transcribed using SuperScript IV First-Strand Synthesis System Kit (Thermo Fisher). The primer sequences are shown in Additional file 2: Table S2. Gene expressions were assessed using a Light Cycler 96 (Roche, Basel, Switzerland). The ΔCt method was used to analyze data, using rp49 as the reference gene. The relative expression values were calculated using the following formula: ΔCt = Ct (target gene)—Ct (reference gene), and the relative expression was equal to 2^−ΔΔCt.