Minibest plant rna extration kit

RNA extraction and cDNA synthesis: Total RNA was extracted from the frozen leaf samples with the MiniBEST Plant RNA Extration Kit (TaKaRa, China). The NanoDrop ND1000 spectrophotometer (Thermo, USA) was used to calculate the RNA concentration and assess purity. The RNA samples with a 260/280 nm absorbance ratio of 1.8–2.0 were retained for further analyses. The RNA integrity was evaluated by 1% agarose gel electrophoresis. The HiScript@III RT SuperMix for qPCR (Vazyme) was used to synthesize cDNA.The qRT-PCR assay was completed with SYBR qPCR Master Mix (Vazyme) and The LightCycle 480 Instrument II(Roche). The reaction solution consisted of 5 μL SYBR qPCR Master Mix (Vazyme), 4 μL cDNA (100 ng), 0.5 μL 10 µM forward primer, 0.5 μL 10 µM reverse primer for a final volume of 10 μL (Table 1). The amplification conditions were as follows: 95°C for 3min s; 40 cycles of 95°C for 10 s, 58°C for 10 s, and 72°C for 25 s, followed by a melting curve analysis from 60 to 95°C. The gene expression levels for each sample were determined based on three replicates.

Total RNA was extracted from the frozen leaf samples with the MiniBEST Plant RNA Extration Kit (TaKaRa, Dalian, China). The NanoDrop ND1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) was used to calculate the RNA concentration and assess purity. The RNA samples with a 260/280 nm absorbance ratio of 1.8–2.0 were retained for further analyses. The RNA integrity was evaluated by 1% agarose gel electrophoresis. The PrimeScript II First Strand cDNA Synthesis Kit (TaKaRa) was used to synthesize cDNA.