Plant rna extraction kit

The ginger samples were ground into powder using liquid nitrogen, and the total RNA was extracted using a Plant RNA Extraction Kit (Vazyme, Nanjing, China) according to the manufacturer’s instruction. Then, the qualified RNA was used for cDNA synthesis using the Vazyme PrimeScript RT reagent kit (Vazyme, Nanjing, China). RT-qPCR was conducted on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, United States). Each reaction contained 5 μl of 2× Maxima SYBR Green qPCR Master Mix (Vazyme, Nanjing, China), 2.5 μl of diluted cDNA (400 ng) template, and 10 μM of gene-specific primers; then, nuclease free water was added to 20 μl. The average threshold cycle was obtained to evaluate the relative expression levels using the 2^–ΔΔCt method. The primer sequences of the MYB genes were adopted from Li et al. (2020) (link), and other genes’ primer sequences were designed by Primer Premier 5.0 (PREMIER Biosoft International, Palo Alto, CA, United States). The primers sequences used in this study are listed in Supplementary Table 1. Three biological and technical replicates were used for each sample along with a template-free control to check for any contamination.

Total RNA was isolated from 100 mg of leaves (fresh-weight) using a plant RNA extraction kit (Nanjing Vazyme Biotech Co., Ltd.). cDNA synthesis was performed with the Evo M-MLV Mix Kit with gDNA Clean for qPCR AG11728 [Accurate Biotechnology (Hunan) Co., Ltd.]. Each reaction contained 10 μl of 2*SYBR Green Pro Taq HS Premix AG11701 [Accurate Biotechnology (Hunan) Co., Ltd.], 2 μl of template cDNA, and 0.4 μl of each forward and reverse primers (10 μM). The GAPDH gene was used as an internal reference. All primers used in this assay are shown in Supplementary Table S1. The PCR reaction procedure was performed as follows: incubation at 95°C for 2 min followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. Each gene was tested in biological triplicates with three technical repeats. The expression level for each sample was expressed as 2^–ΔΔCT. The data were exhibited as the mean ± SD of three independent experiments.

Total RNA was isolated from 100 mg of R. glutinosa roots (fresh-weight) using a plant RNA extraction kit (Nanjing Vazyme Biotech Co., Ltd.). The cDNA synthesis was performed with the Evo M-MLV Mix Kit with gDNA Clean for qRT-PCR AG11728 (Accurate biotechnology (Hunan) Co., Ltd.). Each reaction contained 10 µL of 2 × SYBR Green Pro Taq HS Premix AG11701 (Accurate Biotechnology (Hunan) Co., Ltd.), 2 µL of template cDNA and 0.4 µL of each forward and reverse primers (10 µM). The data were normalized on the basis of the 18S rRNA (DQ469606) threshold cycle (Ct) value. All primers used in this experiment are shown in Table S1d. The qRT-PCR reaction procedure was performed as follows, incubation at 95°C for 2 min followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. Each gene was tested in triplicates with three technical repeats. The expression level for each sample was expressed by the 2^−△△CT method (Livak and Schmittgen, 2001 (link)). The data were exhibited as the mean ± SD of three independent experiments.