Sybr green fluorescent qpcr master mix

Total RNA was extracted from leaf samples taken at 7, 14, 21, and 28 days after exposure to SYST2 VOCs using TRIzol reagent (Invitrogen Biotechnology Co., Carlsbad, CA, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized using reverse transcriptase (TaKaRa Bio Inc., Tokyo, Japan) and random hexamer primers. Real-time PCR was performed with SYBR Green/Fluorescent qPCR master mix (Takara) on a Roche-480 system (Roche) using the EF-1α gene (Berberich et al., 1995 ) as an internal reference.
The relative expression levels of Exp2, Exp9, Exp18. ACO-1. GA (20ox-1). SlIAA1, SlIAA3, and SlCKX1 were assayed. For qRT-PCR, the instrument was programmed for denaturation at 95°C for 1 min, followed by 40 cycles of amplification at 95°C for 5 s, 57°C for 30 s, and 72°C for 30 s. The specific primers for the target genes and the internal reference gene (EF-1α) are given in Supplementary Table S1. Each sample was replicated three times and the data was analyzed using the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)).

Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) following the instructions of the manufacturer. Primer oligo (dT)₁₈ was used to reversely transcribe 2 μg of total RNA into cDNA using AMV reverse transcriptase (Takara) in RT-PCR. The expression pattern of PDI-V was analyzed using a SYBR Green/Fluorescent qPCR master mix (Takara) on a Roche-480 system (Roche), and the wheat Tubulin gene was used as the internal control. The qRT-PCR program started with denaturation at 95 °C for 1 min, followed by 40 amplification cycles programmed as 95 °C 5 sec, 57 °C 30 sec, and 72 °C 30 sec. The CT values for target and standard control genes were retrieved and the comparative threshold 2^−∆∆CT method was applied to quantify the relative expression of given genes53 (link). All reactions were conducted in three biological replicates with three technical repeats for each replication. Double distilled H₂O was used as template for negative controls. All primers used in the study are listed in Supplementary Table S2.

Total RNA was extracted from leaf samples using TRIzol reagent (Invitrogen Biotechnology Co., Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. To determine the effect of SYST2-VOCs on transcription of genes involved in growth regulation, samples were taken at 7, 14 and 21 days exposure to VOCs while to determine the effect of VOCs on transcription of resistance related genes, samples were taken at 3, 6 and 9 days after inoculation of the pathogen. First-strand cDNA was synthesized using reverse transcriptase (TaKaRa Bio Inc., Tokyo, Japan) and random hexamer primers. Real-time PCR was performed using SYBR Green/Fluorescent qPCR master mix (Takara) on a Roche-480 system (Roche) using the EF-1α gene [52 (link)] as an internal reference. The transcriptional expression levels of resistance related genes RRS1,Pr1a and Pr1b1 while growth regulation-related genes (NtEXPA1, NtEXPA2, and ACO1) were detected. The qRT-PCR program consisted of denaturation at 95 °C for 1 min, followed by 40 amplification cycles at 95 °C for 5 s, 57 °C for 30 s, and 72 °C for 30 s. The specific primers used in this study are listed in Additional file 1: Table S1. Each sample was replicated thrice for qPCR, and 2 − ΔΔCt method was used to analyze gene expression level [53 (link)].