Viia7 real time system

Total RNAs were extracted from whole seedling plants using TRIzol (Invitrogen). DNase I enzyme (Invitrogen) was treated for 30 minutes at 37°C to eliminate contaminated genomic DNA from total RNAs. Five micrograms of total RNA were used for synthesis of first strand cDNA using random primers and then used for real-time qRT-PCR analyses using ViiA 7 real time system (Life Technologies). Reactions were carried out in a total volume of 10 ul with Maxima SYBR green master mix (Thermo Fisher Scientific, USA). Primer sequences used in quantitative RT-PCR analyses are listed in Supplementary Table 1.

After treatments, total RNA from BMDMs was isolated using TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. RNA was quantified using a spectrophotometer (NanoDrop), and cDNA was synthesized from 1 μg of total RNA using the high-capacity cDNA reverse transcription kit with RNAse inhibitor (Invitrogen). SYBR Select Master Mix (Applied Biosystems) was used for quantitative real-time polymerase chain reaction. Reactions were performed using 2 μL cDNA and 100 nmol/L of each of the reverse and forward primers. The following primers were used for quantitative real-time polymerase chain reaction: NOS2 forward: 5′-CAC AGT GTC GCT GGT TTG AA-3′; NOS2 reverse: 5′-TCT CCG TGG GGC TTG TAG TT-3′; Arg1 forward: 5′-TGG ACC CTG GGG AAC ACT AT-3′; Arg1 reverse: 5′-GTA GCC GGG GTG AAT ACT GG-3′; Tgfb1 forward: 5′-CTT TGT ACA ACA GCA CCC GC-3′; Tgfb1 reverse: 5′-TAG ATT GCG TTG TTG CGG TC-3′; Gapdh forward: 5′-AGT GCC AGC CTC GTC TCA TA-3′; Gapdh reverse: 5′-GAC TGT GCC GTT GAA CTT GC-3′. Reactions were performed in a ViiA7 Real-Time System (ThermoFisher Scientific). Gapdh was used as an internal control to calculate relative mRNA levels.