Mirneasy mini kit

HUVECs (2 × 10⁶), in the young, intermediate age, or senescence phase, were grown in 100 mm culture dish for 24 h, at 37 °C, in 5% CO₂. Next, the medium was removed, and cells were washed twice with PBS. Culture medium was replaced with fresh medium supplemented with EV-depleted fetal bovine serum (GIBCO Exosome Depleted FBS), 10 µM Gb3, or 25 nM Lyso-Gb3, or 60 µM (TBH), and cells were cultured for 24 h. Next, conditioned medium (CM) was collected for exosome isolation, as above described. In brief, CM was centrifuged at 300× g for 10 min, then at 2000× g for 20 min, and finally at 10,000× g for 30 min. The supernatant was ultracentrifuged, using a SW 28 Swinging-Bucket Rotor (Beckman Coulter, Brea, CA, USA) at 110,000× g, for 70 min. The sEVs pellet was washed in phosphate-buffered saline (PBS), to eliminate contaminating proteins, and centrifuged again at 110,000× g, for 70 min. The final pellets containing the sEVs were re-suspended in PBS, and RNA extraction was carried out with miRNeasy^® Mini kit (Qiagen, cat. No. 217004), according to standard protocol. After removing the CM, adherent cells were washed twice with PBS and detached with trypsin-EDTA. Then, 1 × 10⁶ cells were collected and processed for intracellular RNA extraction with an miRNeasy^® Mini kit (Qiagen, cat. No. 217004), according to standard protocol.

200 μl of serum were used to extract total RNA, including small RNAs, by using miRNeasy Mini kit (Qiagen) according to the supplementary protocol provided for miRNA isolation from serum. A 5-μl aliquot of 5 nmol/l Syn-cel-miRNA-39 miScript miRNA Mimic was spiked into each sample before nucleic acid preparation to monitor the efficiency of miRNA recovery and to normalize miRNA expression in the subsequent real-time PCR analyses^{52 (link)}.
Total RNA, including small RNAs, was also extracted by miRNeasy Mini kit (Qiagen) from liver tissues homogenized by polytron. The RNA was spectrophotometrically quantifed by NanoDrop 2000c, ThermoScientific.

Total RNA was extracted using both TRIzol (Invitrogen, Carlsbad, CA, United States) and the miRNeasy Mini Kit (QIAGEN, Valencia, CA, United States) according to the manufacturers’ protocols, with some modifications. Each sample was resuspended in 300 μl TRIzol reagent (Invitrogen). Resuspension was homogenized for 30 s using a Kontes Pellet Pestle Cordless Motor (Sigma-Aldrich, St. Louis, MO, United States) and placed on ice for 30 s, the steps being repeated 5 times for full homogenization. Then, an additional 700 μl TRIzol was added to the samples and incubated at room temperature for 5 min. 1-bromo-3-chloropropane (200 μl, Sigma-Aldrich) was then added to each sample, followed by incubation for 3 min. Next, the samples were centrifuged at 13,499 × g for 15 min at 4°C using a centrifuge (Smart R17 plus, Hanil Science, Incheon, South Korea). The supernatant (approximately 750 μl) of each sample was mixed with 750 μl of 100% isopropanol. The mixed samples were incubated at -20°C for 2 h. The columns of miRNeasy Mini Kit (QIAGEN) were additionally used to purify the small RNA-enriched RNA and clean RNA according to the manufacturer’s protocol.

mRNA and miRNA was isolated together using miRNeasy mini kit (Qiagen). cDNA for mRNA quantification was prepared from 1 ug of RNA using the High–capacity cDNA reverse transcription kit (Applied Biosystems) with random primers. mRNA cDNA was then diluted 1:5 in water and 4 ul was used for real time PCR. For quantitation of KSHV-pre-miRNAs, RNA was isolated using the miRNeasy mini kit (Qiagen). 1 ug of RNA was then DNase treated using the TURBO DNA-free kit (Life technologies) and used for cDNA synthesis via the High–capacity cDNA reverse transcription kit (Applied Biosystems) with random primers. cDNA was diluted 1:5 before real time PCR. For quantitation of mature miRNAs (human and viral), 30–40 ng of RNA was reverse transcribed with specific RT primers using the MicroRNA Reverse Transcription Kit (Applied Biosystems). The primer-specific cDNA for miRNAs was then diluted 1:6.66 in water.

Approximately 10 mg of cell pellets were frozen in liquid nitrogen immediately after centrifugation at 8000×g for 10 min at 4 °C, and cell walls were broken by liquid nitrogen mortar grinding. Cell pellets were re-suspended in TRIzol reagent (Ambion, Austin, TX) and mixed well by vortexing. Total RNA extraction was achieved using a miRNeasy Mini Kit (Qiagen, Valencia, CA). Contaminating DNA in RNA samples was removed with DNase I according to the instructions for the miRNeasy Mini Kit (Qiagen, Valencia, CA). The RNA quality and quantity were determined using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA) and subjected to complementary DNA (cDNA) synthesis. The RNA integrity number (RIN) of every RNA sample used for sequencing was more than 7.0. To enrich small RNA for the sRNA-seq analysis, the pool of total RNAs was size-selected, and transcripts smaller than 250 nucleotides (nt) was used to prepare cDNA libraries. For each sample, 500 ng size-fractionated sRNAs were subjected to cDNA synthesis using a NuGEN OvationW Prokaryotic sRNA-Seq System according to the manufacturer’s protocol (NuGEN, San Carlos, CA). The resulting double-stranded cDNA was purified using the MinElute Reaction Cleanup Kit (Qiagen, Valencia, CA).