Maxima sybr green qpcr master mixes

Total RNA of specimens and cells harvested and washed with PBS were extracted with TRIzol reagent (Invitrogen). cDNA was then reverse transcribed with oligodT using M-MLV reverse transcriptase (Thermo Fisher Scientific, Rockford, IL, USA). Quantitative real-time PCR was performed with Maxima SYBR Green qPCR Master Mixes (Thermo Fisher Scientific) in an ABI 7300 system (Applied Biosystem, Foster City, CA, USA). The relative expression levels of genes of interest were determined using the 2−ΔΔCt method, GAPDH served as an internal control. The primers used are listed in Supplementary Table 3.

Total RNA was extracted using a TRIzol Plus RNA Purification kit (Invitrogen, Life Technologies), and then treated with RNase-Free DNase I (Thermo Scientific) to remove potential genomic DNA contamination. The cDNA synthesis was carried out using a SuperScript VILO cDNA Synthesis Kit (Invitrogen, Life Technologies). Quantitative real-time PCR was performed on an ABI StepOnePlus Real-Time PCR System (Applied Biosystems) using SYBR Green PCR Master Mix (Applied Biosystems) or Maxima SYBR Green qPCR Master Mixes (Thermo Scientific). The cycling conditions were set according to the instrument’s instructions. The CT values were normalized to the corresponding UBQ10 gene (At4g05320). The primer sequences used are listed in S9 Table.

Total RNA of specimens and cells harvested and washed with PBS was extracted with TRIzol reagent (Invitrogen). Samples with 260/280 values of 1.8‐2.0 were considered for further research. cDNA was then reverse transcribed with oligodT using M‐MLV reverse transcriptase (Thermo Fisher Scientific). Quantitative real‐time PCR was performed with Maxima SYBR Green qPCR Master Mixes (Thermo Fisher Scientific) in an ABI 7300 system (Applied Biosystem). The relative expression levels of genes of interest were determined using the 2−ΔΔCt method, and GAPDH served as an internal control. The primers used are listed in Table S1.

RNA was isolated with Trizol and converted to cDNA with High-Capacity cDNA Reverse Transcription Kit. Quantitative real-time PCR (qPCR) was performed with Maxima SYBR Green qPCR Master Mixes (Thermo Scientific) as described. Primers for Arg1 (QT00134288) were purchased from Qiagen. Each analysis was performed triplicate and normalized to β–actin.

High quality intact RNA samples were processed for cDNA synthesis. First, 1 μg of each sample was DNase I (Fermentas, Thermo Fisher) treated and the DNA-free RNA was reverse transcribed according to the instructions in the Revert Aid H Minus cDNA Synthesis Kit (Thermo Fisher Scientific). Oligo-(dT)₁₈ primers were used for cDNA synthesis. cDNA reaction mixtures were diluted (1.5–5 ng template per reaction) and used in the qPCR reactions together with 250 nM of each primer and the Maxima SYBR Green qPCR Master Mixes (Thermo Fisher Scientific). Reactions were set up in 20 μl volume and run following the manufacturer’s instructions with the inclusion of melt curve analysis in the end of the run. qPCRs were performed in a StepOnePlus thermal cycler (Applied Biosystems, Carlsbad, CA, United States). All primers (Sigma-Aldrich) used in the qPCRs are listed in Supplementary Table S1. The gene encoding tryptophanyl-tRNA-synthetase (TtRNA) (GL50803_3032) was used as an endogenous control in the qPCR reactions (Einarsson et al., 2016b (link)). The fold change in gene expression was calculated using the ΔΔCt method. Significant changes in RNA levels between treatments and controls were assessed using one-way analysis of variance (ANOVA) at α < 0.05 followed by the Dunnett’s multiple comparison test at P < 0.05.