Primerscript 1st strand cdna synthesis kit

The total RNA was obtained from four weeks old seedlings. Samples of fresh roots and shoots were collected using Trizol reagent (Life, Japan). For each sample, approximately 2μ of RNA was used for reverse transcription with a PrimerScript 1st strand cDNA synthesis Kit (Takara, Japan). The QRT-PCR assays were carried out using SYBR Premix Ex Taq (Takara, Dalian, China) on the AB17500 PCR system (Life Technologies, USA). The expression levels of target genes were normalized to that of OsActin. All QRT-PCR assays were performed in three independent replications. Relative gene expression levels were detected using the 2^–ΔΔCT method [20 (link)]. The primers used in this study are listed in Table 1.

Total RNA of all samples was extracted using an RNAprep Pure Plant kit (Tiangen, Beijing, China), and approximately 1 μg RNA was used for the synthesis of first-strand cDNAs with PrimerScript 1st Strand cDNA synthesis kit (TaKaRa, Dalian, China) according to the manufacturer protocol. The gene-specific primers were designed, according to the CDSs of cotton GPAT genes, using Primer v5.0 software. The specific primers used are listed in Table S2, and GhUBQ7 was used as an internal control to normalize all data. The qRT-PCR was strictly conducted with SYBR premix Ex Taq Kit (TakaRa) following the manufacturer's instruction using ABI 7500 real-time PCR System (Applied Biosystems, Foster City, CA, USA). Each sample was conducted with three biological replicates and three technical replicates. The relative expression levels of GhGPAT genes were calculated using the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)).

The total RNA of all samples was extracted using TRIzol reagent (Invitrogen, CA, USA) following the manufactures’ instructions, and approximately 1 µg RNA was reverse transcribed with PrimerScript 1st Strand cDNA synthesis kit (TaKaRa, Shiga, Japan) and miRcute Plus miRNA cDNA T (Tingen, Beijing, China) for genes and miRNAs, respectively, according to the manufacturer protocol. qRT-RCR assays for TaGH9 and miRNAs were conducted on an Eco Real-time PCR system (Illumina, CA, USA) using TB Green Premix Ex Taq (Tli RNaseH Plus) kit (TaKaRa, Shiga, Japan) and SYBR^® miRcute Plus miRNA PreMix (Tingen, Beijing, China), respectively, as described previously [28 (link),75 (link)]. Wheat 18S gene served as the reference gene for TaGH9 analysis, and U6 was used as a reference gene for miRNA analysis. The relative expression levels were calculated according to the 2^–ΔΔCt method for relative expression with three biological replicates and three technical replicates [76 (link)]. The primers used in this study were designed using the Primer premier 5.0 (Primer, CAN, UK) program and are listed in Table S4.

Total RNAs of all the collected samples were extracted using EASYspin Plus RNAprep Kit (Aidlab, Beijing, China). The quantity and quality were determined by a NanoDrop 2000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). First-strand cDNA was synthesized with PrimerScript 1st Strand cDNA synthesis kit (TaKaRa, Dalian, China). All the protocols followed to the manufacturer's instructions. qRT-PCR was performed with Lightcycler 96 system (Roche, Mannheim, Germany) using SYBR the premix Ex taq (TakaRa, Dalian, China) in 20 μL volume according to the supplier's protocols. The specific primers used were listed in Supplementary Table 1, and cotton UBQ7 was used as an internal control. Three biological replicates were performed for each sample. The relative expression levels were calculated according to the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)). The heatmap for expression profiles were generated with the Mev 4.0 software (Saeed et al., 2003 (link)).

G. barbadense cv. 7,124 seeds were cultivated in commercial soil at 28 °C with a photoperiod of 16 h light/8 h dark. Two-week-old seedlings were gently uprooted, rinsed and cultivated in Hoagland solution for two days. Then these seedlings were treated with Hoagland solution containing 0.1 mM MeJA and 1 mM SA, respectively. Roots were sampled from three biological replicates after treatment for 1, 2, 6, and 12 h, then immediately immersed in liquid nitrogen and stored at −80 °C for qPCR.
The total RNA from the root samples treated with MeJA and SA and the control (roots from Hoagland solution without hormone) was extracted using Trizol (Invitrogen). The first strand cDNA was generated from 1 µg of total RNA using a PrimerScript™ 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). qPCR was performed with three replicates using an ABI QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) and SYBR Premix Ex Taq™ (Takara, Dalian, China). The amplification procedure was as follows: one cycle at 95 °C for 3 min; then 40 cycles at 95 °C for 15 s, 60 °C for 15 s. The cotton actin (AF059484) was selected as the internal reference gene (Zhang et al., 2013 (link)). Gene expression levels were calculated according to the 2^−ΔΔCT method described by Livak & Schmittgen (2001) (link). The primers used for qPCR are listed in Table S1.

Using a miRNeasy mini kit (Qiagen, Hilden, Germany), RNA was isolated from the M2 macrophages and subjected to qRT-PCR. Primers for arg1, il-10, il-4, tgf-β, il-12p40, ifn-γ, and β-actin were obtained from Bioneer Inc. (Daejeon, Korea). cDNA was synthesized using PrimerScript 1st strand cDNA Synthesis Kit (Takara, Shiga, Japan). qPCR using SYBRGreen (Qiagen) was conducted on a real time cycler (Bio-Rad, Hercules, CA). According to the Livak method 29 (link), data for relative expression were analyzed.