Circulating MicroRNAs: Methods and Protocols by Nobuyoshi Kosaka
By Nobuyoshi Kosaka
microRNAs (miRNAs) are small non-coding RNAs that keep an eye on quite a few organic phenomena, comparable to improvement and homeostasis. The dysregulation of miRNA results in affliction development, fairly of melanoma. In Circulating MicroRNAs: equipment and Protocols, specialist researchers within the box aspect contemporary advances within the isolation, purification and research of circulating miRNAs from quite a few resources for study. The publication is split into 3 major themes. the 1st part contains the research of secretory miRNAs in cell-cell verbal exchange, and the second one, the examine of circulating miRNAs in physique fluids. The final describes the radical thoughts used to review circulating miRNAs. Written within the hugely winning Methods in Molecular Biology™ sequence structure, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, with no trouble reproducible laboratory protocols, and key pointers on troubleshooting and keeping off recognized pitfalls.
Authoritative and practical, Circulating MicroRNAs: tools and Protocols seeks to assist scientists in facing the new advances of RNAi expertise from the bench to the bedside.
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Adjust final volume to 50 ml with D2O. 2 μm syringe filter. Keep the solution in the fridge. 12. S. , Del Mar, CA, USA), or similar model. 13. Magnetic stir plate. 14. Peristaltic pump. 15. NanoDrop 2000c spectrophotometer (Thermo Scientific). 16. 0 software (NanoSight). 17. Refractometer. 18. RNase A. 19. TRIzol® Reagent (Invitrogen). 20. 8 %, for ACS analysis, stabilized with ethanol. 21. miRNAeasy Mini Kit (Qiagen). 22. Qiacube (Qiagen), optional, for fully automated sample processing using Qiagen spin columns.
4. The following day, the exosomes are transferred to a sterile Ultra-Clear™ (14 × 89 mm) Beckman tube, and pelleted by centrifugation (100,000 × g, 60 min, 4 °C). 5. Remove the supernatant very gently, leaving the exosome pellet with approximately 100–200 μl of liquid in the tube. 5 ml RNase-free microcentrifuge tube, and keep it on ice. Measure the amount of exosomes (based on their protein content) with a NanoDrop 2000c spectrophotometer. The exosome size and concentration can be measured by nanoparticle tracking analysis with a NanoSight’s instrument (NanoSight) (Fig.
B) Nanoparticle tracking analysis, with a NanoSight instrument, of size (histogram) and concentration (table) of exosomes purified from culture supernatants of BMDCs. (c) Analysis, by detection of the exosome marker CD9 by Western blot, of each 1-ml-density fraction and pellet of a continuous sucrose gradient used to purify BMDC-derived exosomes. Exosomes, detected by their CD9 content, are enriched in those fractions with characteristic exosome density 30 Angela Montecalvo et al. spin-bar into the chamber closer to the gradient maker outlet.