Primescript 2 first strand cdna synthesis kit

Among all differentially expressed genes, 10 genes identified by RNA-seq analysis were selected for validation by qRT-PCR. Samples preparation procedure was the same as described in Section 4.5. Total RNA was extracted using TRIzol, and then cDNA was synthesized from total RNA using PrimeScriptTM II First Strand cDNA Synthesis Kit (Takara, Dalian, China) according to the manufacturer’s instructions.
All qRT-PCR experiments were performed in three technical replicates using GAPDH as the reference gene. The qRT-PCR primers used in this study were described in Table 1. The cycle conditions were 95 °C for 5 min, 40 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 15 s, and the melting curve temperature is 72~95 °C. Gene expression was calculated using the 2^−∆∆t relative expression method.

Total RNA was isolated from TM-1 seedlings using the RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (Tiangen, Beijing, China). The RNA concentration was determined using the NanoDrop 2000 microvolume spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States), whereas RNA integrity was assessed by 1% agarose gel electrophoresis. First-strand cDNA was synthesized from 1 μg RNA using the PrimeScript^TM II first Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). To clone the vacuolar chloride channel gene in upland cotton, the BlastP and tBlastN programs were performed against the G. hirsutum genome database¹ using the A. thaliana CLCg amino acid sequence as the query. The coding sequences of gene IDs GH_A06G0574 and GH_D06G0541 as the best match were retrieved and gene-specific primers were designed. Full-length cDNAs were amplified using the GhCLCg-1F/R primer pair (Supplementary Table 1) and the KOD-Plus-Neo DNA polymerase (TaKaRa, Dalian, China). The PCR products were purified using the FastPure Gel DNA Extraction Mini Kit (Vazemy Biotech Co., Ltd.) and then cloned using the pEASY^®-Blunt Cloning Kit for the subsequent transformation of Trans1-T1 Escherichia coli competent cells (TransGen Biotech). Positive clones cultured at 37°C were analyzed by sequencing to verify they were correctly transformed.

qRT-PCR was used to confirm the expression of common differentially expressed genes in Pak-choi. Total RNA was extracted from each sample. The first-strand cDNA was synthesized, using a PrimeScript^TM II First Strand cDNA synthesis kit (TaKaRa Bio, Dalian, China), according to the manufacturer’s protocol. The primers were designed, using the software Beacon Designer 7.9, and listed in Table 5. The quantified expression levels of the tested genes were normalized against the housekeeping genes Cyclophilin 1 (CYP1) (Ma et al., 2016 (link)). The qRT-PCR assays were performed with three biological and technical replicates. Each reaction was performed in 20-μl reaction mixtures containing a diluted cDNA sample as template, SYBR Premix Ex Taq (2×) (TaKaRa, Kyoto, Japan) and gene-specific primers. Conditions for quantitative analysis were as follows: 95°C for 3 min, then 40 cycles (95°C 30 s, 60°C 30 s), and 72°C for 30 s. qRT-PCR was performed according to a previous report (Song et al., 2016 (link)). The comparative Ct value method was adopted to analyze the relative gene expression according to a previous analysis and RNA expression levels relative to the actin gene were calculated as 2^–ΔΔCT (Pfaffl, 2001 ; Song et al., 2016 (link)).