Column plant total rna kit

Following the manufacturer’s instructions, 100 mg of B. cusia samples were used to extract total RNA using the Column Plant Total RNA Kit (TransGen, Beijing, China). RNA samples were tested for quality and concentration using ethidium bromide-stained agarose gel electrophoresis and spectrophotometer measurement on a NanoDrop 2000C Spectrophotometer (Thermo Scientific, Waltham, MA, USA). For cDNA synthesis, samples having an optical density absorption ratio at OD260/280 between 1.9 and 2.2 and an OD260/230 more than 2.0 were employed. TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China) was used to create first-strand cDNA from 1 μg of total RNA, according to the manufacturer’s instructions.

The frozen samples were ground to a fine powder in liquid N₂ using a pestle and mortar. The total RNA was extracted from all plant tissue samples using a Column Plant Total RNA Kit (TransGen Biotech, China) following the manufacturer's recommendations. The concentration of RNA samples was determined using a NanoDrop 2000 spectrophotometer (Thermo, America) at 260 nm, whereas its purity was assessed based on absorbance ratios at 260/280 nm. Samples with an optical density absorption ratio at OD260/280 between 1.9 and 2.2 and OD260/230 <2.0 were used for cDNA synthesis. The integrity of purified RNA was confirmed using agarose gel electrophoresis and ethidium bromide staining. Genomic DNA was isolated from young leaves (100 mg) using the cetyltrimethyl ammonium bromide method and checked by agarose electrophoresis. First-strand cDNAs were synthesized using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, China), by adding Oligo (dT) primer, gRemover, E-mix, and R-mix to 1 μg of total RNA. RNase-free water was added to the prior mixture, and the total volume (20 μL) was incubated at 42°C for 15 min according to the manufacturer's protocol. Reverse transcriptase was inactivated by incubating the mixture at 85°C for 5 min, and the cDNA solution was stored at −20°C.