By Nobuyoshi Kosaka
microRNAs (miRNAs) are small non-coding RNAs that keep watch over quite a few organic phenomena, similar to improvement and homeostasis. The dysregulation of miRNA results in affliction development, rather of melanoma. In Circulating MicroRNAs: tools and Protocols, professional researchers within the box aspect fresh advances within the isolation, purification and research of circulating miRNAs from quite a few resources for study. The ebook is split into 3 major subject matters. 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 check circulating miRNAs. Written within the hugely winning Methods in Molecular Biology™ sequence layout, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, easily reproducible laboratory protocols, and key pointers on troubleshooting and fending off identified pitfalls.
Authoritative and practical, Circulating MicroRNAs: tools and Protocols seeks to assist scientists in facing the new advances of RNAi know-how from the bench to the bedside.
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Extra resources for Circulating MicroRNAs: Methods and Protocols
Stop when the gradient reaches 1 cm from the top of the tube. 5. 2), directly add the exosomes (up to 1 ml) on top of the gradient. 2), the volume of liquid containing the exosomes must be reduced (to 1 ml or less), to be able to load the exosomes on top of the continuous sucrose gradient. To do that, centrifuge the exosomes (100,000 × g, 60 min, 4 °C) and resuspend the exosome pellet in 1 ml (or less) of PBS (see Note 12). 6. Centrifuge the continuous gradient overnight (100,000 × g, 16 h, 4 °C) in an SW41 swinging-bucket rotor (brakes set on low).
To stop the lysis, wash the cells twice with cold culture medium. 6. Count viable cells and adjust concentration to 107 cells/ml. 7. Plate 20–22 × 106 BM cells per 175 cm2 culture flask in a final volume of 50 ml of complete culture medium supplemented with mouse GM-CSF (1,000 U/ml) and IL-4 (500 U/ml) (day 1). 8. On day 3 of culture, 40 ml of culture medium (containing floating cells) are removed and centrifuged in 50 ml conical tubes. The cells are resuspended with a similar volume of fresh medium with GM-CSF plus IL-4 and placed back in the flasks.
They are enriched in tetraspanins (CD81, CD63, CD9), cytoskeletal proteins (actin, tubulin), and specific lipids such as phosphatidylserine, ceramide, and cholesterol. However, exosomes also contain specific proteins depending on the nature of the producer cell. So, for example, exosomes from Carolina Villarroya-Beltri and Cristina Gutiérrez-Vázquez contributed equally to this work. ), Circulating MicroRNAs: Methods and Protocols, Methods in Molecular Biology, vol. 1007/978-1-62703-453-1_4, © Springer Science+Business Media New York 2013 41 42 Carolina Villarroya-Beltri et al.
Circulating MicroRNAs: Methods and Protocols by Nobuyoshi Kosaka