Quantitative reverse transcription polymerase chain reaction

Total RNAs were isolated from animals of mixed stages, with 50-μl pellet animals (>200 animals) resuspended in 250 μl of lysis buffer of Quick-RNA MiniPrep kit (R1055, Zymo Research) and subsequently lysed by TissueRuptor (Motor unit “8” for 1 min). Total RNAs were extracted following the manufacturer’s instructions (R1055, Zymo Research). Two micrograms of RNA per sample was reverse-transcribed into complementary DNA (B24408, BioTools). Real-time PCR was performed by using Roche LightCycler 96 and SYBR Green (FERK1081, Thermo Fisher Scientific) as a double-stranded DNA–specific binding dye. qRT-PCR condition was set to 95°C for denaturation, followed by 45 cycles of 10 s at 95°C, 10 s at 60°C, and 20 s at 72°C. Melting curve analysis was performed after the final cycle to examine the specificity of primers in each reaction. Gene expression changes were calculated by the ΔΔCt method, with act-3 used as the reference gene. Primer sequences are listed in table S2.

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