Plant rna kit

To detect the relative expression levels of MtGA20ox, MtGA3ox, MtGA2ox, MtGID1, and MtDELLA genes in root, shoot, leaf, flower, stem, and pod, total RNA was extracted from these tissues on two-month-old wild-type plants. To detect the effects of GA₃ and PAC on the expression of MtGA20ox, MtGA3ox, and MtGA2ox, total RNA of leaves was extracted from plants after 24 h GA₃ and PAC treatment. To detect the relative expression levels of MtGA20ox1, MtCYCB, MtCYCD, and MtKPR, total RNA of leaves was extracted from two-month-old wild-type and overexpression plants.
Total RNA was extracted using a Plant RNA Kit (TransGene Biotech, Beijing, China) following the manufacturer’s instructions. The concentration and quality of the extracted RNA were evaluated using Nanodrop 2000 Spectrophotometer (Thermo Scientific, USA). Reverse transcription of RNA to cDNA was performed with 1 μg total RNA using an iScript cDNA Synthesis Kit (Bio-Rad, Richmond, CA, USA). qRT-PCR was carried out in triplicate for each sample on a CFX Connect™ Detection System (Bio-Rad, Richmond, CA, USA) using TaKaRa SYBR Premix Kit (TaKaRa, Japan). MtUBI gene was used as the internal reference gene. The relative expression levels of the genes were calculated using the 2^-ΔΔCT method. Primer sequences used for qRT-PCR analysis were listed in Table S3.

Total RNA was isolated from plant tissue samples using the Plant RNA Kit (Transgen, Beijing, China). The first strand cDNA synthesis was performed according to the manufacturer’s directions (Thermo Fisher Scientific, Waltham, MA, United States). The quantitative real time PCR (qRT-PCR) was carried out using the CFX96 (Bio-Rad, Hercules, CA, United States) with 20 μL volume (containing 1 μL of 1:10 diluted cDNA, 200 nM of each gene-specific primer, and SYBR Green Mix from Bio-Rad). PCR cycling parameters were set as following: 95°C for 5 min, 40 cycles of 10 s at 95°C and 30 s at 60°C, then a final melting curve at 65°C for 5 s. The stably expressed gene NtL25 (ribosomal protein gene) was used as the internal control for data normalization. Expression data was carried out with three biological and technical replicates and calculated using the 2^–ΔΔCt method (Schmittgen and Livak, 2008 (link)). The primers used in this study are listed in Supplementary Table 3.

Total RNA was extracted using a Plant RNA Kit (Transgen, Beijing, China) following the manufacturer’s instructions. The first-strand cDNA was obtained by reverse transcription using cDNA TranScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, Beijing, China). Primers for qRT-PCR assays (Table S4) were designed using the Primer 3.0 website (http://bioinfo.ut.ee/primer3-0.4.0/). qRT-PCR was performed in a 20-μL volume containing 10 μL SYBR Green (Takara, Japan), 0.4 μL ROX II (Takara, Japan), 0.2 μM gene-specific primers, and 5 μL 5× diluted cDNA. The reaction was performed using the ABI 7500 Real Time Thermal Cycler. The expression of ZmActin1 was used as an internal control (Table S4). For qRT-PCR assays, three biological replicates were performed, with three technical replicates per biological replicate.