Total exosome rna isolation kit

DNA and RNA oligonucleotides were synthesized and purified by HPLC (Sangon Biotech Co., China). DNA Hairpins were prepared as monomers at 10 μM in the reaction buffer (20 mM Tris·HCl, 150 mM KCl, 10 mM (NH4)₂SO₄, 2.5 mM MgCl₂, 1% Triton X-100, pH 7.5) using a snap cooling procedure: heating at 95°C for 5 min followed by cooling at 0.5°C min⁻¹ to 25°C, and allowing equilibration at room temperature for 30 min before use. The working concentration of DNA was 5 μM, and the initiator was 10 nM in HCR.
Urine samples from bladder cancer patients were obtained from cancer hospital, Chinese academy of sciences, Hefei. Exosomes were isolated with polyethylene glycol (34 (link)). The exocrine morphology was characterized by transmission electron microscope (JEM-2100). Size distribution of exosomes was analysised by dynamic light scattering (Zetasizer Nano ZSP). RNA was extracted using a Total Exosome RNA Isolation Kit (Invitrogen). This study was approved by the medical ethics committee of Hefei institute of physical sciences, Chinese academy of sciences and also was performed in accordance with the ethical standards.

RNA was isolated from Müller cell pellets using the RNeasy total RNA isolation kit (Qiagen) as per the manufacturer’s instructions. RNA was also isolated from EV for miRNA sequencing and qPCR validation using the Total Exosome RNA Isolation Kit (Invitrogen^™) as per the manufacturer’s instructions. In order to ensure that only EV-encapsulated RNAs were analysed, intact EVs in solution were treated with RNase A enzyme (100 U/mL) (Invitrogen^™, MA, USA) for 15 min at 37°C before the addition of RNase inhibitor (1 U/mL). To confirm that RNA was internalised, an equivalent quantity of EV was pre-treated with a membrane-disrupting detergent (Triton X-100, 0.1% v/v), vortexed briefly, and incubated for 15 min at room temperature prior to the addition of the RNase enzyme. A second control consisting of 1 μg of total cellular RNA recovered from MIO-M1 cells was diluted in an equivalent volume of PBS before receiving the RNase enzyme treatment. Samples were then re-pelleted by ultracentrifugation at 100,000 × g for 90 min at 4°C prior to miRNA isolation. The RNA was purified and analysed using the methods described above. RNA was quantified by a NanoDrop UV spectrophotometer (Nanodrop-1000, Thermo Scientific) before storage at −80°C.

Total exosomal RNA of OPLL or PLL cells was extracted using a Total Exosome RNA Isolation Kit (Invitrogen, USA) according to the manufacturer’s instructions. MiRNA library construction and miRNA sequencing were performed as described previously [42 (link)] to identify the differentially expressed miRNAs derived from OPLL or PLL cell exosomes. Next, target genes of miR-140-5p were predicted by TargetScan (http://www.targetscan.org) and miRDB (http://mirdb.org). The intersection of these two databases was created by Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny).

RNA profiling was performed on lysate of trophozoites, enriched ElVs, and ElVs treated with Triton X-100 and RNase A using the Total Exosome RNA Isolation Kit (Invitrogen) and following the manufacturer’s instructions available at as-sets, https://assets.thermofisher.com/TFS-Assets/LSG/manuals/total_exosome_kit_man.pdf, accessed on 31 May 2022. This kit utilizes AcidPhenol:Chloroform extraction to provide a robust front-end RNA purification step, followed by a final RNA purification over a glass-fiber filter [71 (link)]. RNA concentrations were determined using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). To compare RNA profiles, a 15% denaturing polyacrylamide gel using 8 M urea and 1× TBE was utilized. The samples were loaded in denaturing buffer and run at 100 V in 1× TBE buffer for 3 h. The gel was visualized using a silver staining kit (Fermentas) following the manufacturer’s protocol. To assess the internalization of the RNA cargo within vesicles, isolated ElVs were briefly vortexed after treatment with 0.1% Triton X-100 and subsequently incubated with RNase A (10 mg/mL) for 15 min at 37 °C. After treatment, RNA was purified as indicated above. The GeneRuler™ Ultra Low Range DNA Ladder (Invitrogen) was used as molecular weight standard.