Steadypure plant rna extraction kit

Two long root hair genotypes (W106 (Jingyang60) and W136 (Honggoudou)) and two short root hair genotypes (W90 (Zaoyangmai) and W100 (Shengen)) were selected based on root hair length screening. Among them, W90 is an American variety, while W100, W106, and W136 are Chinese wheat landraces from Fujian, Shanxi, and Henan Province, respectively. Following seed sterilization, germination, and agar medium cultivation, the seed roots were collected on the fourth day of germination and rapidly frozen in liquid nitrogen (three biological replicates for each sample). RNA extraction followed the user manual of the SteadyPure Plant RNA Extraction Kit (Accurate Biology, Qingdao, China). A NanoDrop 2000 (Thermo, Wilmington, NC, USA) was utilized to evaluate the purity and concentration of RNA.

Total RNA was extracted using the SteadyPure Plant RNA Extraction Kit (AG21019 Accurate Biotechnology, Changsha, Hunan, China) as per the manufacturer’s instructions. cDNA was synthesized by reverse transcription using the Evo M-MLV RT Kit (AG11728, Accurate Biotechnology, Changsha, Hunan, China). Real-time quantitative PCR (qRT-PCR) was performed using SYBR^® Green Premix pro Taq HS qPCR Kit (AG11701 Accurate Biotechnology, Changsha, Hunan, China) in a 20 μL reaction volume that contained 2 μL cDNA, 0.4 μL 2 μM forward primer, 0.4 μL 2 μM reverse primer, 0.4 μL ROX Reference Dye (50X), 10 μL 2 × SYBR Green Premix Premix pro Taq, and 6.8 μL RNase-free water. Ten candidate genes in the coumarin biosynthetic pathway were selected for qRT-PCR validation, and the actin gene [13 (link)] was used as an internal reference control gene. The specific primers for the qRT-PCR of the genes are shown in (Supplementary Table S8), and the qPCR amplification conditions were as follows: 95 °C for 30 s; 40 cycles at 95 °C for 5 s; and 60 °C for 30 s. Relative expression was calculated using the 2^−ΔΔCt method [34 (link)], and the results were obtained for each gene using three biological replicates and three technical replicates of each sample.

Total RNA was extracted using the SteadyPure Plant RNA Extraction Kit from Accurate Biotechnology (Hunan) Co., Ltd. The quality and concentration of RNA were evaluated using a micro-volume nucleic acid and protein analyzer and RNase-free agarose gel electrophoresis. After confirming the quality and concentration of RNA, the Evo M-MLV Reverse Transcription Premix Kit from Accurate Biotechnology (Hunan) Co., Ltd. was employed to reverse transcribe it into cDNA following the instructions. The cDNA was then stored at −20°C for future use.

Total RNA was extracted from 100 mg of tree peony buds using a SteadyPure Plant RNA Extraction Kit (Accurate Biotechnology, Hunan, China) according to the manufacturer’s instructions. The first strand of cDNA was synthesized from 1 μg of total RNA using a PrimerScript™ RT Reagent Kit (TaKaRa, Dalian, China). qRT-PCR was performed using a SYBR^® Premix Ex Taq™ II Kit (TaKaRa, Dalian, China). All reactions were performed in triplicate. Relative expression levels were analysed according to Livak and Schmittgen [56 (link)]. The primers used for qRT-PCR are listed in Table S1 (see online supplementary material).

The expression patterns in response to abiotic stresses (PEG6000-induced drought stress and NaCl-induced salt stress) were examined in order to evaluate the role of three IbPGs by using qRT-PCR. All the primers used in this study were designed using the NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and listed in Table S9. Total RNA was extracted from the frozen samples by using SteadyPure Plant RNA Extraction Kit (Accurate Biotechnology, Hunan, China.) in accordance with the manufacturer’s instructions to validate the RNA-seq data. Reverse transcription was performed on 1 µg of RNA from each sample using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (Accurate Biotechnology, Hunan, China). The qRT-PCR assay was conducted by a CFX Connect Real-Time System (Bio-Rad, Veenendaal, UT, USA) using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Hunan, China). The β-Actin gene (GenBank, AY905538) was used as an internal reference to evaluate the relative gene expression level. Three replicates of the tests were carried out, and the data were computed using the 2^-ΔΔCT method [59 (link)]. We analysed the data and compared the means using LS at a 0.01 level of significance.

Total RNA was extracted from the frozen banana plant using SteadyPure Plant RNA Extraction Kit (Accurate Biotechnology Co., Ltd., Hunan, China) following the manufacturer's instructions. HiScript II One Step qRT-PCR SYBR Green kit (Vazyme Biotech, Nanjing, China) was employed for qRT-PCR assays according to the manufacturer's instructions. First-strand cDNA was prepared by reverse transcription from 1 μg of DNA-free total RNA in a final reaction volume of 20 μl. RT-qPCR was conducted using a QuantStudio 5 Real-Time PCR System (Applied Biosystems, CA, USA) in four replicates. The qTUB gene (banana) was used as a reference for data normalization, and the target genes were amplified using the primer sets listed in Supplementary Table S1. The relative transcript abundance of each gene was estimated using the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)).