Rnx plus kit

The RNA content of the MVs was extracted using the RNX Plus™ kit (SinaClon, Tehran, Iran). Due to low amounts of extracted mRNAs, the detection of specific mRNA content of MVs was performed according to SinaClon First Strand cDNA Synthesis Kit (SinaClon, Tehran, Iran) and qPCR Master Mix (A320799 Ampliqon, Denmark). Designed primers for specific mRNAs are depicted in supplementary Table 1. Quality control of the primers was carried out by LinReg PCR software (Amsterdam, Netherlands).

Total RNA was isolated using the RNX‐PLUSKit (SINACLON, Iran) according to the manufacturer's protocol. The number of 3 × 10⁴ cells of YM‐1 or KYSE‐30 cells were cultured in the flask. After 24 h incubation, cells were treated with DMSO (0.3% as control) or entacapone (IC50 = 140 μM) for 48 h. Then, cells were harvest with trypsin solution and washed twice with PBS. To extract the RNA, briefly, cells were disrupted and homogenized with RNX‐PLUS reagent at room temperature (RT) for 5 min. Then, chloroform was added to the samples and shacked vigorously for 5–10 s, then incubated for 15 min. Subsequently, using centrifugation (12 000 rpm at 4°C for 15 min), the upper aqueous phase containing the nucleic acids was isolated. Then, it was precipitated by adding equal volume of isopropanol followed with incubation for 15 min on ice and centrifugation. The RNA was washed twice by adding 1 ml 75% ethanol, and centrifugation at 7500 rpm at 4°C for 8 min. Finally, the purified RNA samples were dissolved in RNase‐free water and stored at −80°C.

According to the manufacturer's instructions, viral RNA was extracted from all samples using the RNX-PLUS kit (Sinaclon, Iran). In brief, 1 ml of RNX-PLUS solution and 200 μl of chloroform were added to the tube containing the sample. Then, the mixture was centrifuged at 12000g at 4°C for 15 min. Next, the aqueous phase was transferred to a new tube, and an equal volume of isopropanol was added. The supernatant was discarded after centrifugation, and 1 ml of 75% ethanol was added to the mixture. Afterward, the supernatant was removed, and the sediment was dried at room temperature. The concentration of the RNAs extracted was measured using a spectrophotometer. Following this, complementary DNA (cDNA) synthesis was performed using SinaClon first-strand cDNA synthesis kit according to manufacturer's instructions.