Aceq qpcr sybr green master mix

Total RNA was extracted from heart, liver, spleen, lung, kidney, pancreas, muscle, mPFC, midbrain, cerebellum, brainstem, amygdala, hippocampus, hypothalamus, hypophysis, spinal cord, medulla oblongata, and olfactory bulb tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as previous described [34 (link)]. RNA purity was determined by the ratio of OD value at 260/280 nm (1.8–2.0). Total RNA was reversely transcribed in a 20 μL reaction mixture at 25°C for 10 min, 42°C for 30 min, and 85°C for 5 min with 5× QRT SuperMix (Vazyme, Nanjing, China). The primer pair sequences for the target genes (Table 1) were generated by Primer Premier 5 software. Real time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in a 20 μL system including 2 μL cDNA (50–100 ng/μL), 10 μL AceQ^™ qPCR SYBR Green Master Mix (TransGen, Beijing, China), 0.4 μL ROX Reference Dye II, 0.8 μL (10 μM) of each primer, and 6 μL RNase-free water. PCR program is the same as previously described [35 (link)]. The qRT-PCR data were analyzed using the 2^−ΔΔCt method for mRNA quantification.

The infected leaves were collected according to the previously described method for RNA-seq sample preparation, and RNA was extracted. A total of 1 μg of RNA per sample was used for reverse transcription using the PrimeScript RT Kit (Takara Biotechnology (Dalian) Co. Ltd., Dalian, China). Then, AceQ^® qPCR SYBR Green Master Mix (TransGen Biotech Co., Ltd., Beijing, China) was used for qRT–PCR assays with a Bio–Rad CFX96 System. The primers employed for qPCR were designed with the NCBI Primer design tool [61 ]. Primer sequences for qRT–PCR are listed in Table S3. Relative expression was calculated using the 2^−ΔΔCt method. Three independent biological replicates were used for each sample.