Plant rna extract kit

Digital tissue expression pattern of GmMYB181 in soybean was examined using SoyBase database¹ and soybean eFP Browser².
Total RNA was extracted from soybean and Arabidopsis with Plant RNA Extract Kit (TianGen, Beijing, China) and cDNA was reverse transcribed using PrimeScript^TM 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). qRT-PCR was conducted with SYBR^® Green Real-time PCR Master Mix (Toyobo, Japan) on a iQ5 real-time PCR instrument (Bio-Rad, United States). SqPCR was carried out with 2 × Taq PCR MasterMix (TaKaRa, Dalian, China). All primer pairs used are listed in Supplementary Tables S1, S2. Soybean internal genes including actin (Glyma.04G215900) and tubulin gene (GenBank Accession No. AY907703) were used for SqPCR and qRT-PCR, respectively. Arabidopsis tubulin gene (AT5G62690) was used as internal control. The relative expression levels of GmMYB181 were calculated utilizing the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)).

Total RNA was extracted using Plant RNA Extract Kit (TianGen, Beijing, China) according to the manufacturer’s instructions and cDNA was synthetized with M-MLV reverse transcriptase (TaKaRa, Dalian, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out with ABI 7500 system (Applied Biosystems, Foster City, CA, USA) using ChamQ™ SYBR qPCR Master Mix (Vazyme, Nanjing, China). The PCR was performed with the following parameters: 94 °C for 1 min and 40 cycles of 95 °C for 15 s, 60 °C for 15 s, 72 °C for 45 s followed by a final extension at 72 °C for 10 min. The relative expression levels of GmFILa were normalized using soybean endogenous gene tubulin (GenBank accession no. AY907703) and were estimated utilizing the 2^-ΔΔCt method [71 (link)]. Semi-quantitative RT-PCR (sqPCR) was conducted with 2 × Hieff™ PCR Master Mix (Yeasen, Shanghai, China), and Arabidopsis tubulin gene (AT5G62690) was chosen as an internal control. The PCR protocol was 95 °C for 5 min and 30 cycles of 94 °C for 30 s, 56 °C for 40 s, 72 °C for 1 min followed by a final extension at 72 °C for 10 min. All the gene-specific primer pairs were listed in Additional file 1: Tables S2 and S3.

Total RNA was isolated from 100 mg of each tissue sample following the instruction of plant RNA Extract Kit (TIANGEN Biotech, Beijing, China). The cDNA was synthesized with the Prime Script RT Master Mix kit (TaKaRa, Shiga, Japan). All cDNA samples were diluted to an equal concentration using the NanoDrop‐2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and used as templates for quantitation in a 20‐μl reaction system with three biological replicates. The GmUKN1 (Glyma.12g020500, GenBank accession no. NM_001254696.2) and AtACTIN7 (AT5G09810, GenBank accession no. NM_121018.4) were chosen as reference genes to normalize the relative expression level of GmSWEET39 in soybean and Arabidopsis, respectively.
The qRT‐PCR was performed using LightCycler480 System (Roche Diagnostics Ltd, Rotkreuz, Switzerland) with SYBR Premix Ex Taq Kit (TaKaRa). The relative expression of GmSWEET39 in Arabidopsis was calculated by 2^‐∆Ct and in soybean by 2^‐∆∆Ct methods (Livak & Schmittgen, 2001). The sequences of all primers are listed in Table S1.