Faststar universal sybr green master rox

Realtime quantitative PCR (qRT-PCR) was carried out using an Applied Biosystems StepOnePlus Real-Time PCR System with FastStar Universal SYBR Green Master (Rox) (Roche) [17 (link)]. The gene primers used in qRT-PCR reactions are listed in S1 Table. For each gene, all PCR reactions were carried out in triplicate within a single plate, with rpoA used as the reference gene. Quantification of relative gene expression was analyzed using the 2^-ΔΔCt method [22 (link)].

Total RNA was isolated with the TransZol Reagent (TransGen, China), and cDNA was synthesized by using TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (TransGen Biotech, Beijing, China) based on the manufacturer’s instructions. The qRT-PCR reactions were performed in 20 μL reactions using 1 μL of cDNA, with the FastStar Universal SYBR Green Master (ROX) (Roche Diagnostics, Indianapolis, IN, USA), on the ABI 7500 system (Thermo Fisher Scientific, Waltham, MA, USA). The qRT-PCR conditions were as described previously [54 (link)]. Three independent biological replicates were conducted. The M. truncatula ACTIN gene (Medtr3g095530) was used as a standard control, and the 2^−ΔΔCT method was used to calculate the relative levels of gene expression. The expression levels in the control plants without treatment (0 h) were normalized to 1. Data were statistically analyzed using Duncan’s test with SPSS19 and different letters indicate statistically significant differences (p < 0.05). The primer sequences used in this study are listed in Table S1.

The tissue samples, such as leaves, were immediately frozen in liquid nitrogen after sampling from the field. The total RNA was extracted with TRIzol reagent (TransGen Biotech, Beijing, China) as per the manufacturer’s instructions. Approximately 2 µg of total RNA was reverse transcribed, and the first strand of cDNA was obtained by reverse transcription by using Superscript III reverse transcriptase kit (Invitrogen, California, USA) as per manufacturer’s instructions. qRT‐PCR experiments were performed using FastStar Universal SYBR Green Master (ROX) (Roche, Basel, Switzerland) kits on an ABI 7500 system (Thermo Fisher Scientific, California, USA). The relative expression was calculated. The rice actin gene was used as an internal reference (Livak and Schmittgen, 2001 (link)). The primers used in the experiment are listed in Table S1.