Lightcycler 96 sw 1

Total RNA was extracted from all samples using the RNAiso Plus Kit (Takara, Kusatsu, Japan) according to the manufacturer’s instructions. Equal concentrations of the RNA were reverse transcribed to cDNA using the TransStart All-in-One first-strand cDNA synthesis supermix (TransGen Biotech, Beijing, China). The cDNA was then used for quantitative real time PCR (qRT-PCR) using the SYBR Green kit (Bio-Rad, Hercules, United States), performed on a LightCycler 96 SW 1.1 instrument, to analyze the transcription levels of gene-silenced strains. Primer sequences for the target genes are listed in Table 1. Expression data for genes in silenced strains were normalized against the internal reference gene 18S rRNA. The relative expression levels were calculated by comparing the cycle threshold (Ct) of each target gene with the corresponding internal reference gene using the 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)).