Dna engine opticon 2 real time pcr detection system

Real-time quantitative PCR (RT-qPCR) was performed to determine the expression levels of differentially expressed genes of A. lancea cultured in vitro. Elongation factor 1 alpha gene (EF1a) was used as an internal reference (Yuan et al., 2016b (link)). Primers for the selected DEGs are listed in Supplementary Table S2. One microgram of total RNA was transcribed into cDNA using HisCRIPT^® II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., Nanjing, China) according to the manufacturer’s instruction. Subsequently, RT-qPCR was conducted using the DNA Engine Opticon 2 Real-time PCR Detection System (Bio-Rad, Hercules, CA, United States). The reaction system consisted of a volume of 20 μL, which included 10 μL of 2 × AceQ qPCR SYBR^® Green Master Mix (High ROX Premixed) (Vazyme Biotech Co., Nanjing, China), 2 μL of the cDNA template, 0.4 μL of each primer, and 7.6 μL ddH₂O. The reaction conditions were 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. All assays were performed in triplicate. Relative expression levels for each cDNA sample were calculated by the 2^−ΔΔCt method (Wang et al., 2015 (link); Liu et al., 2018 (link)).

The specific primer pairs were designed for the qRT-PCR of each GLO gene (Additional file 8). Total RNA was purified from rice using TRIzol® reagent (Life Technologies, Carlsbad, USA), and further treated with DNase I (RNase free, Toyobo, Osaka, Japan). The quality of the isolated RNA was assessed with a NanoDrop-1000 (Thermo Fisher Scientific, Bremen, Germany). One microgram of RNA was used as a template for first-strand cDNA synthesis using ReverTra Ace (Toyobo, Osaka, Japan). The qRT-PCR reaction mixture consisted of 0.2 μM (each) primer, 10 μL of 2 × SYBR Green PCR Master Mix (Toyobo, Osaka, Japan), and 2 μL of appropriate diluted cDNA. The analysis was conducted using a DNA Engine Opticon 2 Real-Time PCR Detection system and Opticon Monitor software (Bio-Rad, Hercules, CA). The data were normalized to the amplification of the OsActin1 gene (Os03g0718100).

Cells were mock-infected or infected with Ad-GFP, Ad-ARID2, AdR-siARID2, or AdR-sicontrol. Thirty-six hours after infection, total RNA was isolated using TRIzol (Invitrogen, Rockville, MD) following the manufacturer's instructions, as previously described [31 (link)]. RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Promega) in the presence of random hexamers (Promega). PCR-based amplification of the respective genes was carried out using the cDNA products as templates. SYBR Green-based qPCR analysis was carried out using the DNA Engine Opticon 2 real-time PCR detection system (Bio-Rad, CA, USA). Relative expression was calculated as a ratio of the expression of the specific transcript to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Each sample was analyzed in triplicate. The primer sequences used for PCR are listed in Supplementary Table 2.