Direct zoltm rna miniprep

Total RNA was extracted from hippocampal tissue using Direct-zolTM RNA MiniPrep (Zymo Research, Tustin, CA, USA) according to the manufacturer’s instructions, and quantified by measuring the absorbance at 260 nm. RNA was amplified using the MyGo Mini Real-Time PCR system (IT-IS Life Science, Ltd., Cork, Ireland). One-step RT-qPCR was performed using the MyGo Green 1-step Low Rox (IT-IS Life Science, Ltd.) for a total volume of 20 µL and a template concentration of 10 pg/µL total RNA, according to the manufacturer’s recommendations. The thermal cycling conditions were 45 °C for 10 min as an RT step and 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 20 s. An automatic melting temperature analysis was performed to ensure a single amplified product at the end of the reaction (melting from 60 °C from 95 °C at a ramp rate of 0.1 °C/s). The relative quantification (fold change) of mRNA expression was estimated by the use of the 2^−ΔΔCt method [32 (link)] as follows: Relative mRNA expression was defined as 2^−Δ(ΔCt), where ΔCt = Ct_TARGET – Ct_{Housekeeping gene} and Δ(ΔCt) = ΔCt_treated groups – ΔCt_{CS group}, and Ct_{Housekeeping gene} is the average of the Ct values of β-actin, which was used as the housekeeping gene for each sample to normalize the targeted gene expression.

Following the manufacturer’s technique, the Trizol reagent was used to extract total RNA from the liver and brain tissues (Direct-zolTM RNA MiniPrep, catalog no. R2050 Zymo, Irvine, CA, USA). The purity of RNA samples (1 µL) was verified by measuring their absorbance using a NanoDrop spectrophotometer (ND-1000, Thermo Scientific, Foster City, CA, USA) at 260 and 280 nm, and RNA ratios (A260:A280) greater than 1.8 were used for further experiments. cDNA was obtained from each sample following the manufacturer’s instructions (SensiFastTM cDNA synthesis kit, Bioline, catalog No. Bio-65053 Bioline GmbH, Luckenwalde, Germany).
The reaction mixture was composed of up to 1 μg of total RNA and contained 20 μL of DNase-free water, 4 μL of 5× Trans Amp buffer, 1 μL of reverse transcriptase, and 20 μL of DNase-free water. The final reaction mixture was then put into a thermal cycler and subjected to the following process: primer annealing at 25 °C for 10 min, reverse transcription at 42 °C for 15 min, and inactivation at 85 °C for 5 min. The samples were then stored 4 °C.

Total RNA was extracted from E. multilocularis primary cell samples after 24 h in culture (Fig 1) using Direct-zol^TM RNA MiniPrep (ZYMO RESEARCH) according to the manufacturer's protocol. To maximize precipitation of small RNA, the eluted RNA was incubated overnight at -20°C with the addition of 0.1 volumes of 3 M sodium acetate (pH 5.2), 2.5 volumes of ethanol and 2 μl of glycogen (10mg/ml). RNA was centrifuged at 14,000 g for 30 min at 4°C, washed in 75% ethanol, air dried at room temperature and resuspended in 20 μl of nuclease-free water. Samples were stored at -80°C until use. RNA concentration was determined using a Nanodrop 1000 spectrophotometer (Thermo Scientific) and RNA integrity was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies) using a RNA 6000 Nano chip.