Quantstudio 1 real time pcr system

Levels of MC-producing Microcystis in water and aerosol samples were quantified with Quantstudio 1 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) by targeting the mcyE gene that encodes Microcystis-specific MC production with the set of primers and PCR conditions from a previous study (Sipari et al., 2010 (link)). PCR reaction solutions contained TOPreal™ qPCR 2x PreMIX with SYBR green (Enzynomics, Daejeon, South Korea), 10 μM of each primer and the extracted DNA (total volume was 20 μL). The thermal cycling conditions were 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 30 s, and 62°C for 1 min, and by a melting curve stage of 95°C for 15 s and 60°C for 1 min (Lee et al., 2021 (link)). The output data were analyzed by associated software (Design and Analysis Software 2.6.0, Applied Biosystems, CA, USA). All experiments were performed in triplicate.

RNA was extracted from the aortic tissue of mice in the acute model or from VSMCs using the RNeasy Mini Kit (Qiagen, Valencia, United States). Complementary diribonucleic acid (cDNA) was synthesized from 1.0 μg of extracted total RNA using ReverTra Ace (TOYOBO, Osaka, Japan). The synthesized cDNAs were subjected to polymerase chain reaction (PCR) using the TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, United States) and predesigned gene-specific primer and probe sets (TaqMan Gene Expression Assays; Applied Biosystems). TaqMan gene expression probe-and-primer sets for PPARα, superoxide dismutases (SOD1 and SOD2), nicotinamide adenine dinucleotide phosphate oxidases (NOX2 and NOX4), CAT, heme oxygenase-1 (HO-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), and transforming growth factor-β1 (TGF-β1) were purchased from Applied Biosystems. Real-time PCR was performed in triplicate for each sample using the QuantStudio 1 real-time PCR System (Applied Biosystems) described previously (42 (link)). The results were quantified using the relative Ct method and normalized to the internal control, glyceraldehyde 3-phosphate dehydrogenase. See Supplementary Table 1 for PCR primer details.

DNA‐free RNA was obtained using the Rneasy Plus Micro Kit (QIAGEN) with DNase treatment, and total RNA was reverse transcribed using a Hiscript II Q RT SuperMix for qPCR (+gDNA wiper) Kit (Vazyme, Nanjing, China). Real‐time PCR was performed in triplicate using ChamQ Universal SYBR qPCR Master Mix and the QuantStudio1 Real‐Time PCR System (Applied Biosystems, Foster City, CA) following the manufacturer's protocol. Gene expression was normalized relative to the gene expression levels of hypoxanthine guanine phosphoribosyl transferase (Hprt). The following primers are shown in Table S11 (Supporting Information).

RNA was extracted from the cells using the RiboEXTM reagent (GeneAll, Republic of Korea) according to the manufacturer's instructions. The extracted RNA was converted to cDNA using the SmartGene compact cDNA Synthesis kit (SMART GENE, Republic of Korea). Real-time PCR was performed using the TOPrealTM SYBR Green qPCR PreMIX (Enzynomics, Republic of Korea) on a QuantStudio™ 1 Real-Time PCR system (Applied Biosystems, USA). The expression levels of the target genes were normalized to the housekeeping gene beta-actin, and the fold change was calculated using the 2^-ΔΔCT method [21 (link)]. The primer sequence will be provided upon request.

Total RNA was extracted from frozen liver (~ 20 mg) using a RNeasy Plus Universal Mini Kit (QIAGEN), as per manufacturer’s instructions. The concentration of eluted RNA was measured using a Nanodrop DN-1000 spectrophotometer. cDNA synthesis was carried out using the qScript synthesis kit, following the manufacturer’s protocol (Quantabio). mRNA expression was measured by quantitative (Q)-PCR using SYBR Green Mastermix (Eurogentec Ltd.) and the DNA Engine Opticon 2 system (BioRad). Primers were obtained from QuantiTech Primer Assay (QIAGEN) and product details are as follows: Rn_Hk2_1 QT00190764, Rn_Pfkl_1 QT00175651, Rn_Ldha_2 QT02336243, Rn_Higd1a_1 QT00372428, Rn_Stoml2_1 QT01571724 , Rn_Slc25a11_1 QT01082914 , Rn_Actb_1 QT00193473. In the instance of Cox7a2l, QuantiFast SYBR Green PCR kit (QIAGEN) and QuantStudio 1 Real-Time PCR System (Applied Biosystems, Thermo Fisher) were used. The primer was obtained from QuantiTect Primer Assays (QIAGEN) with the following product details: Rn_Cox7a2l_1_SG. In all cases, transcript levels were normalised to levels of Actb and fold change determined using the 2^−ΔΔCT method, with expression in vehicle/normoxic animals normalised to 1.

RNA was isolated using an RNA-Xpress^TM reagent according to the manufacturer’s protocol, and the reverse transcription reaction foresaw the incubation of 1 μg of total RNA from each sample with All-in-One 5X RT Master Mix (Abcam, Cambridge, UK). qPCR was performed using the QuantStudio 1 Real-Time PCR System (Applied Biosystems, Foster City, CA,USA) and TaqMan Gene Expression Master Mix (Applied Biosystems, CA, USA). The qPCR TaqMan probes (Applied Biosystems, Foster City, CA, USA) were as follows: Hs00174114 m1_IL2; Hs00907777_m1 IL2RA; IL-1β: Hs01555410_m1; TNF: Hs00174128_m1; and IL-6: Hs00174131_m1. β-actin was used as a housekeeping gene, all experiments were carried out in triplicate, and the ΔΔCt method was used to quantify the relative expression of all the cytokines tested.

Samples were processed for total RNA extraction using TRIzol™ Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The 500 ng of isolated total RNA was used for cDNA synthesis using AccuPower^® RT PreMix (Bioneer, Daejeon, Korea) in accordance with manufacturer’s instructions. qRT-PCR was performed using a QuantStudio 1 Real-Time PCR system (Applied Biosystems, Waltham, CA, USA) with reaction conditions as follows; 50 °C for 10 min, 95 °C for 10 min, 95 °C for 30 s, and 60 °C for 30 s (40 cycles), followed by melting curve analysis. The GAPDH gene was used as a housekeeping gene, and relative gene expression level was calculated using the 2^−ΔΔC_t method [19 (link)]. To confirm intestinal marker gene expression, gel electrophoresis was performed in this study. In brief, the qRT-PCR products were mixed with Dyne LoadingSTAR (Dyne Bio Inc., Seoul, Korea) and were run in gel electrophoresis at 100 V for 30 min by using Agaro-Power™ System (Bioneer, Daejeon, Korea). The primer information used in this study is listed in Table 1.