Maglisto 5m cell total rna extraction kit

The expression levels of the efflux pump genes acrB and oqxB and their regulator genes rarA, ramA, soxS, and marA were assessed by qRT‐PCR. Total RNA of CRKP isolates was extracted with the MagListo^™ 5M Cell Total RNA Extraction Kit (Bioneer, Daejeon, Korea) according to the manufacturer's instructions. Reverse transcription of RNA to cDNA was performed using AccuPower^® CycleScript RT Premix (Bioneer, Daejeon, Korea). Quantitative real‐time PCR performance using Solg^™ 2X Real‐Time PCR Smart mix with EvaGreen^™ 500 (SolGent Co., Ltd., Daejeon, Korea) was run on a Exicycler^™ 96 Real‐time Quantitative Thermal Block (Bioneer, Daejeon, Korea). All experiments were performed in triplicate. The mRNA expression level of each gene was normalized based on an endogenous reference gene (rrsE). Relative expression of each gene was calculated based on tigecycline‐susceptible isolate CRKP 87 (tigecycline MIC 0.5 mg/L, expression = 1) as the control strain. The level of expression of each gene was calculated using 2^−ΔΔCt method. The primers used in the experiments are listed in the Table S2.

The organoids (Passage 4) were treated with Cell Recovery Solution (Corning Incorporated, Corning, NY, USA) to remove the Matrigel, and total RNA was extracted using the MagListo™ 5 M Cell Total RNA Extraction Kit (Bioneer, Daejeon, Republic of Korea) according to the manufacturer's guidelines. Subsequently, the RNA product was subjected to a quantitative reverse transcription-polymerase chain reaction (RT-PCR). Firstly, RNA was synthesized to complementary DNA (cDNA) using AccuPower® RocketScript™ Cycle RT Premix (Bioneer). Quantitative polymerase chain reaction was performed by mixing the cDNA with AccuPower® 2X Greenstar qPCR Master Mix (Bioneer). The mRNA expression level was measured with Thermal Cycler Dice® Real Time System III (Takara, Shiga, Japan). The primer sequences are given in Table 2. The negative control (no cDNA template) was ran simultaneously with every assay, and PCR from each cDNA sample was ran in triplicate. Constitutively expressed GAPDH was used as an endogenous control to correct any potential variations in RNA loading or reaction efficiency. The results were presented as relative fold changes using the GAPDH reference and applying the formula 2^-ΔΔCt. The PCR products were loaded into 1% agarose gel and visualized with a DNA gel staining solution under UV light.

Total RNA was extracted from isolated tissues or organoids using MagListo™ 5 M Cell Total RNA Extraction Kit (Bioneer, Daejeon Metropolitan City, Korea) following the manufacturer’s protocol. Thereafter, 1 μg of RNA was used to synthesize cDNA using PrimeScript™ RT Master Mix (TaKaRa, Kyoto City, Japan). Quantitative RT-PCR was performed with a Thermal Cycler Dice® Real-Time System III (TaKaRa, Kyoto City, Japan) using SYBR® Premix Ex Taq™ II (TaKaRa). The PCR primers sequences are listed in Supplementary Table 2. PCR experiments were carried out in triplicates.