Nanodrop spectrophotometry

Cardiac tissues were removed, submerged in liquid nitrogen, and kept at −70 °C until further analysis. The extraction of RNAs was performed by RNX^TM reagent according to the manufacturer’s procedure (Sina Clon Bioscience, Tehran, Iran). Concentration of RNA, and its purity were calculated by measuring the ratio of 260/280 nm optical density using Nanodrop spectrophotometry (Eppendorf, cologne, Germany), and values between 1.8–2 were defined as an acceptable purity. The cDNA synthesis was performed using qPCR^TM Green Master Kit for SYBR Green I^® (Yekta Tajhiz, Tehran, Iran) according to the instructions of the manufacturer. Real-time PCR was performed in Roche Light-Cycler detection system (Basel, Switzerland) with the following steps: Initial denaturation for 5 min at 95 °C and 45 cycles of denaturation for 15 s at 95 °C, annealing for 30 s at 60 °C, an extension for 20 s at 72 °C followed by melt curve analysis (50–99 °C) [40 (link)]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene to measure relative gene expression. The results were evaluated by using 2^−ΔΔCt comparative method and Light Cycler SW1.1 software. The sequence of the primers is shown in Table 2.

The total RNA was extracted from HeLa cells overexpressing of eGFP, eGFP::Hs‐CDC45, eGFP::Mm‐CDC45, eGFP::Cr‐CDC45 post‐transfection 48 h using RNAiso plus reagent (Takara). The integrity and quality of total RNA was determined by 1% agarose gel electrophoresis and Nanodrop spectrophotometry (Eppendorf). Each sample collection was repeated three times. Transcriptome sequencing was performed by Novogene. The library was constructed followed NEB common library construction method.⁵⁸ Illumina sequencing was operated after qualified of examination in depot. The clean reads were obtained for subsequent analysis after filtration of original data, error rate examination of sequencing and examination of GC content distribution. The clean reads were then compared to the reference genome. Differential gene enrichment was analyzed using NovoMagic (https://magic.novogene.com/).

Total RNA was extracted from ovaries using TriPure total RNA isolation kit according to the manufacturer’s procedure (Roche Molecular System, USA), dissolved in dimethyl pyrocarbonate treated water and quantified at a wavelength of 260 nm by nanodrop spectrophotometry (Eppendorf, Hamburg, Germany). The RNA with optical density absorption ratio OD260 nm/OD280 nm between 1.8 and 2.0 was used for reverse transcription (RT) reaction. Genomic DNA was removed by treating 1 μg of isolated RNA with 2 units of DNase I (Fermentas Inc, Vilnius, Lithuania).

After supernatant collection, the remaining cells in experimental plates were extracted for viral RNAs using TRIzol reagent (Invitrogen, California, USA) according to the manufacturer’s protocol. The samples were loaded into the Direct-zol™ RNA MiniPrep (Zymo research, California, USA) and quantified by Nanodrop spectrophotometry (Eppendorf, New York, USA). The RT-qPCR was performed with a Step-OnePlus Real-Time PCR System (Applied Biosystems, California, USA.) with 1 × Power SYBRGreen PCR Master Mix, 400 nM each of capsid primers^{40 (link)} or 250 nM of NS1 primers^{41 (link)} and 0.1 µg of total RNA. The reactions were then cycled at 48 °C 30 min and 95 °C 10 min, followed by 45 cycles of 95 °C for 20 s (denaturation), 55 °C for 30 s (annealing), 72 °C for 30 s (extension). Each sample was analyzed in triplicated and results were confirmed by two independent experiments.