Evo m mlv mix kit with gdna clean for qpcr ag11728

Total RNA was isolated from 100 mg of leaves (fresh-weight) using a plant RNA extraction kit (Nanjing Vazyme Biotech Co., Ltd.). cDNA synthesis was performed with the Evo M-MLV Mix Kit with gDNA Clean for qPCR AG11728 [Accurate Biotechnology (Hunan) Co., Ltd.]. Each reaction contained 10 μl of 2*SYBR Green Pro Taq HS Premix AG11701 [Accurate Biotechnology (Hunan) Co., Ltd.], 2 μl of template cDNA, and 0.4 μl of each forward and reverse primers (10 μM). The GAPDH gene was used as an internal reference. All primers used in this assay are shown in Supplementary Table S1. The PCR reaction procedure was performed as follows: incubation at 95°C for 2 min followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. Each gene was tested in biological triplicates with three technical repeats. The expression level for each sample was expressed as 2^–ΔΔCT. The data were exhibited as the mean ± SD of three independent experiments.

Total RNA was extracted from IPEC-J2 cells (1 × 10⁷) according to the method described in section “2.3 RNA-sequencing (RNA-seq) and data analysis.” Cytoplasmic and nuclear RNAs of IPEC-J2 cells were isolated using a PARIS™ Kit reagent kit (Ambion, Austin, TX, USA) according to the instructions provided by the supplier. Total RNA was reverse transcribed into cDNA using Evo M-MLV Mix Kit with gDNA Clean for qPCR AG11728 (Accurate Biotechnology, Hunan, China). The qRT-PCR assay was performed according to the instructions of SYBR Green assay (Accurate Biotechnology, Hunan, China) and performed on a Roche LightCycler 480 (Roche Applied Science, Mannheim, Germany). Gene expression levels were normalized to that encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to determine the relative expression using the 2^–ΔΔCt method (Ljvak, 2001 (link)). The PCR primers for transcripts and genes are listed in Table 1.

Total RNA was isolated from 100 mg (fresh-weight) of leaves using the plant RNA extraction kit (Nanjing Vazyme Biotech Co., Ltd.) and synthesis of cDNA was performed with Evo M-MLV Mix Kit with gDNA Clean for qPCR AG11728 (Accurate biotechnology (Hunan) Co., Ltd.). Each reaction contained 10 μl of 2xSYBR Green Pro Taq HS Premix AG11701 (Accurate biotechnology (Hunan) Co., Ltd.), 2 μl of template cDNA, 0.4 μl of forward and reverse primers each (10 μM). GAPDH gene was used as an internal reference (Table 1). The PCR reaction procedure was as follows, incubation at 95°C for 2 min followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Each gene was tested in three biological replicates, with three technical repeats. The expression level for each sample was expressed as 2^–ΔΔCt. The data were exhibited as the mean ± SD of three independent experiments.