Ecotm real time pcr system

Maxima First Strand complementary DNA (cDNA) synthesis kit (Thermo Fisher Scientific Inc., San Diego, CA, USA) was used to convert the total RNA (1 µg) into cDNA, which were then loaded into a thermal cycler (Labnet, USA). The cDNA was then used to quantify the selected gene expressions (Table 1) on the Eco^TM Real-Time PCR System (Illumina Inc., San Diego, CA, USA) using a Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific Inc., San Diego, CA, USA). The mixture was prepared by mixing 12.5 µL of Maxima SYBR Green qPCR Master Mix, 0.3 µM of forward and reverse primers and ≤ 500 ng of cDNA. Nuclease-free water was used to top up the mixture up to 25 µL. The mixture was then run using the Eco^TM Real-Time PCR System (Illumina Inc., San Diego, CA, USA) platform and the reaction was set at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15s, and annealing/extension at 55–60 °C for 15 to 30s.

The total RNA was extracted using Trizol TRI Reagent Solution (ThermoScientific) according to the manufacturer’s standard protocol, cDNA was synthesized according to the manufacturer’s instructions from the total RNA using the reverse transcription BioFact^TM RT Kit. The real-time polymerase chain reaction was conducted using an Eco^TM real-time PCR system (Illumina) using 2× Real-Time PCR Master Mix, including SYBR Green I (Biofact) and 10 pmol of each primer, processed in a two-step PCR program. All analyses were conducted in triplicate, and gene expression data were analyzed using ECO Study program (Illumina). Relative expression levels were determined by testing treated and control plants, and normalized to GmActin.

Expression analysis of Actin, TaSAMS, TaDMAS1, TaYSL15, TaPCS1. and TaMT1 was performed by quantitative qRT-PCR (reverse transcription PCR). Briefly, tissues (50–100 mg) were ground with a mortar and pestle to a fine powder in liquid nitrogen. Afterward, total RNA was isolated as instructed by SV Total RNA Isolation System (cat. no. Z3100), Promega Corporation, USA. The integrity of isolated RNA was then checked by denaturing agarose gel electrophoresis and quantified by NanoDrop 2000 UV-Vis Spectrophotometer. The first-strand cDNA was then synthesized by using GoScript^TM Reverse Transcription System (Cat no. A5001), Promega Corporation, USA. Before real-time analysis, the cDNA samples were treated with RNAase for removing RNA contamination. Real-time PCR was performed in triplicate using GoTaq^®qPCR Master Mix (Promega USA) and gene-specific primers (Supplementary Table S1) in an Eco^TM real-time PCR system (Illumina, USA). Expression data was normalized with Actin as an internal control (Eco Software v4.0.7.0). The real-time PCR program used was as follows: 3 min at 95°C, 40 cycles of 30 s at 94°C, 15 s at 58°C, and 30 s at 72°C.