7300 system sds software v1

Stem-loop RT-PCR was used to verify the expression levels of miRNAs between the two strains (Tang et al., 2006 (link)). Briefly, total RNA from embryos, instar larvae, pupae, male and female adults was extracted by RNAiso Plus reagent, respectively. 2 μg total RNA was reverse-transcribed to cDNA using AMV transcriptase (TaKaRa, Dalian, China) and looped antisense primer. The mixture was incubated at 42°C for 60 minutes and 85 °C for 5 minutes. qRT-PCR was performed on the ABI Prism 7300 HT Sequence Detection system (Applied Biosystem, CA, USA) using FastStart® SYBR Green (Applied Biosystem, CA, USA). Reactions were incubated in a 96-well optical plate at 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1min. A melting curve program was run immediately after the PCR and the data was analyzed by 7300 System SDS Software v1.2.1 (Applied Biosystems). Table S2 was the primers for the stem-loop quantitative RT-PCR. The raw threshold cycle (Ct) values were normalized against U6 standard to obtain normalized Ct values, which were used to calculate relative expression levels in samples using the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)).