Sybr fast universal qpcr mix

Total RNA (1 μg) was reverse-transcribed into first-strand cDNA using a PrimeScript^TM RT reagent kit (Takara Bio, Inc., Otsu, Shiga, Japan). Quantitative PCR (qPCR) was performed using a 10 μL reaction containing 1 μL cDNA, 0.3 μL each of 10 μmol⋅L^–1 forward and reverse primers (Supplementary Table S1), and 5 μL SYBR FAST Universal qPCR mix (KAPA Biosystems, Woburn, MA, United States) in a LightCycler 480 system (Roche Diagnostics GmbH, Mannheim, Germany), with the following amplification conditions: 5 min at 95°C, followed by 45 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 20 s. The qPCR experiments were performed for each sample using three biological and three technical replicates. The expression levels of selected genes were normalized to the expression levels of D. citri actin-1. The differential gene expression was calculated using the 2^–△△Ct method (Livak and Schmittgen, 2001 (link)), and the results were expressed as mean ± SE. The differences in gene expression levels between infected and uninfected D. citri were analyzed using t-tests in the SPSS 18.0 statistical software (P < 0.01).

The qPCR primer for examined AAAP genes was designed using Primer Premier (v5.0) software (Premier Biosoft, Rockville, MD, USA) (Table S4). To ensure the specific amplification of the intended target genes in BPHs, all qPCR primers used in the following study were firstly checked using the online software tool of Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast) prior to qPCR experiments [47 (link)] (checked on 15 February 2021). The qPCR system of the 10 μL reaction sample consisted of 1 μL cDNA, 0.3 μL forward and reverse primers (concentration of 10 μmol/L), 5 μL SYBR FAST Universal qPCR mix (KAPA Biosystems, Woburn, MA, USA), and 3.4 μL ddH₂O. qPCR assays were conducted using a Light Cycler 480 System (Roche Diagnostics, Basel, Switzerland) and the SYBR FAST Universal qPCR Kit (KAPA Biosystems, Woburn, MA, USA), with a PCR amplification procedure as follows: one cycle at 95 °C for 5 min, followed by 45 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s. For the entire experiment, three biological replicates and three technical replicates were used for each treatment.

Total RNA (1 μg) was used to synthesize first-strand cDNA using the PrimeScript^TM RT reagent kit (Takara Bio, Inc., Otsu, Shiga, Japan). The qPCR assays were performed using a Light Cycler 480 System (Roche Diagnostics, Basel, Switzerland) and the SYBR^®, FAST Universal qPCR Kit (KAPA, Woburn, MA, United States) following the manufacturer’s instructions. A 10 μL reaction mixture containing 1 μL cDNA, 0.3 μL each of 10 μmol/L forward and reverse primers, and 5 μL SYBR^®, FAST Universal qPCR mix (KAPA Biosystems, Woburn, MA, United States) was prepared. The PCR amplification conditions were as follows: 5 min at 95°C, followed by 45 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 20 s. Three biological and three technical replicates for each sample were performed. Gene expression levels were normalized to the expression levels of BPH β-actin (Chen et al., 2013 (link)). Changes in gene expression were calculated using the 2^–△△Ct method (Livak and Schmittgen, 2001 (link)), and the results are expressed as mean ± SE. The differences in gene expression levels between treatments were analyzed using the Student’s t-test in the SPSS 18.0 statistical software (P < 0.05). In this study, the expression profile of NlAPC09 in the ovaries of BPHs from two types of populations (described above) was determined, and the primers used for qPCR experiments are listed in Supplementary Table 1.