Sybr green pro taq hs kit

Total RNA was prepared from the serum, AML‐12 cells or mice liver tissues adopting Steadypure Universal RNA Extraction Kit (Accurate Biotechnology) separately, followed by the production of cDNA utilizing Evo M‐MLV RT Mix Kit (Accurate Biotechnology). PCR amplification was performed on the CFX Opus 96 Real‐Time PCR system (IMH‐Bio) via the SYBR® Green Pro Taq HS kit (Accurate Biotechnology). The calculation of relative gene expression was achieved by virtue of the 2^−ΔΔCq method. GAPDH expression was used for normalization.

Patients (≥18 years old; IA, n = 12) who were diagnosed with IA at Xiangya Hospital of Central South University were recruited for this research between December 2021 and June 2022. Patients with other diseases or complications were excluded. In total, 12 healthy controls who were gender- and age-matched were also selected.
Peripheral blood monocytes (PBMCs) were isolated from the blood samples of IA and healthy persons using Ficoll solution (Solarbio, Beijing, China). Total RNA was extracted from the PBMCs using TRIzol reagent (Takara, Kyoto, Japan) according to the manufacturer’s protocol. Then, reverse transcription reactions were performed using 500 ng RNA and an Evo M-MLV reverse transcriptase kit (Accurate Biotechnology, Hunan, China) according to the manufacturer’s instructions. A SYBR Green Pro Taq HS Kit (Accurate Biotechnology, Hunan, China) and 0.4 μmol of each primer pair were used to amplify the cDNA, which was then evaluated in an ABI 7500 real-time PCR system (Applied Bioscience, Foster City, CA, United States). The results were analyzed using the 2^–ΔΔCt method and expressed as the ratio of the internal control, GAPDH. The primer sequences used for RT–qPCR are available in Supplementary Table 1.

The transcript levels of IbNCED1 in leaf, stem and root tissues of the 4-week-old in vitro-grown plants and leaflet, leaf, stem, pencil root, and storage root tissues of the 80-day-old field-grown plants of Jishu26 were analyzed with qRT-PCR using SYBR Green Pro Taq HS kit (AG11701, ACCURATE BIOLOGY). Furthermore, the 4-week-old Jishu26 plants were stressed in Hoagland solution with 100 mM ABA and 100 mM GA, respectively, and sampled at 0, 3, 6, 12, and 24 h after stresses for analyzing the expression of IbNCED1. Ibactin (AY905538) was used to normalize the expression levels in sweet potato [71 (link)]. All the specific primers are shown in Supplementary Table S2.

Using TRIzon Reagent, total RNA was isolated from cells and EVs (CWBio, China). SYBR Green Pro Taq HS Kit was used to evaluate the expression of microRNA and mRNA (Accurate Biology, Jinan, China). The reverse transcription of RNA to cDNA was followed by Evo M-MLV RT Premix for qPCR (Accurate Biology, Jinan, China). The GAPDH gene was utilized as a reference. As internal control and external reference for miRNA expression in cells and EVs, respectively, U6 and cel-miR-39-3p (GenePharma, Shanghai, China) were selected. Using the 2Ct technique, the relative expression of miR-2467-5p and other genes was determined. The sequences of mRNA primers are provided in Additional file 17: Table S5.

Quantitative real‐time polymerase chain reaction (qRT‐PCR) reagents and supplies are AG RNAex Pro RNA kit, SYBR Green Pro Taq HS kit, Evo M‐MLV reverse‐transcription kit (removal gDNA reagent) and Rox and were purchased from Accurate Biotechnology (Hunan) Co. Ltd. People's Republic of China. Western‐blotting reagents and supplies are Rabbit Anti‐Ngb, Polyclonal Antibody (bs‐1859R), Rabbit Anti‐HIF‐1, Alpha Polyclonal Antibody (bs‐0737R), Rabbit Anti‐beta‐Actin (Loading Control), Polyclonal antibody (bs‐0737R) and goat anti‐rabbit IgG/HRP(bs‐0295G‐HRP) and were purchased from Bioss Co. Ltd. People's Republic of China. RIPA tissue or cell rapid lysate was purchased from Bio topped, and 0.22μm polyvinylidene difluoride filter (PVDF) membranes, 4 × protein loading buffer (DTT), Rainbow 245 broad‐spectrum protein marker (11‐245KD) and ECL hypersensitivity luminescent solution were purchased from Solarbio Co. Ltd. People's Republic of China. Immunohistochemical reagents and supplies are immunohistochemical staining kit and HRP‐DAB kit and were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd. People's Republic of China.