Sybr green real time rt pcr

Rotor gene-6000 (Corbett, USA) was used to measure the gene expression of these proteins. Pairs of primers for each gene were designed using Primer 3 software, synthesized by Bioneer (Bioneer, Germany), and worked with a final concentration of 100 nm. The primer sequences are presented in Table 2. For this purpose, the kit (SYBR-green Real-Time RT-PCR, TAKARA, Japan) was used. According to the kit instructions, a 20 μl amplification reaction was performed, consisting of 10 μl of the original solution (Master Mix), 0.3 μl of the Forward primer, 0.3 μl of the Reverse primer, two μl of the synthesized cDNA, and 7.4 μl of distilled water. The PCR temperature pattern for the respective genes was 95°C for 10 min for the first cycle, which continued with 45 cycles in two 95°C stages for 20 seconds and 60°C for 60 seconds. To analyze the data, first, the ΔCt of the gene in each sample was calculated as the difference between Ct of the corresponding gene and GAPDH gene as a reference gene ^{16 (link)}. Reactions were performed in triplicate, and expression levels were calculated using the CT comparative method (2−ΔΔCT) ^{5 (link)}.

In Experiment 4, the total RNA was extracted from shoots and roots of plants using the Total RNA Extraction Kit (TIANGEN, China). The complementary DNA (cDNA) was synthesized in accordance with Fast Quant RT Super MixReverse Transcription Kit instructions (TransGene, Beijing, China). Quantitative real-time PCR was performed using SYBR Green Real-time RT-PCR (Takara) and an ABI7500 Fast Real-Time PCR System (Applied Biosystems). The primers used for real-time PCR are shown in Supplementary Table 1. The relative gene expression level was calculated using the 2^–ΔΔCt method. Each real-time PCR experiment contained three technical replicates.

In experiment 3, total RNA was extracted from the shoots and roots of plants using the Total RNA Extraction Kit (TIANGEN, China). The cDNA was synthesized in accordance with Fast Quant RT Super Mix Reverse Transcription Kit instructions (TransGene, Beijing, China). Quantitative real-time PCR was performed using SYBR Green Real-time RT-PCR (Takara) and an ABI7500 Fast Real-Time PCR System (Applied Biosystems). The primers used for real-time PCR are shown in Supplementary Table 1. The relative gene expression level was calculated using the 2^–ΔΔCt method. Each real-time PCR experiment contained three technical replicates.