Saves money - does not require expensive equipment (ultracentrifuge and rotors)
- Saves time – exosome and microvesicle capture and RNA extraction can be completed in minutes instead of hours – days
- Allows fractionation of different types of microparticles
- Even difficult cell free fluids can be processed
- Allows high volumes (up to 60 mL) of cell-free fluids to be processed
- Maximizes RNA recovery
- Ideal for miRNA and mRNA studies of microvesicles and exosomes
- The ExoMir™ PLUS Kit is specifically designed for the collection of samples “in the field” where large-capacity centrifuges are not available
The ExoMir™ Kit is designed for the fractionation of, and RNA extraction from, exosomes and other microparticles in cell-free fluids. The ExoMir Kit uses a rapid fractionation approach in which samples are passed over syringe filters to capture exosomes and larger membrane-bound particles. The filters are then flushed with an RNA extraction reagent to lyse the captured particles and release their contents. In the standard procedure, samples are passed over 2 filters connected in series, to effectively capture particles such as apoptotic bodies, microvesicles on the upper filter and exosomes on the lower filter. Then the filters are disconnected and separately flushed with BiooPure™-MP to lyse the captured particles and release their contents. BiooPure-MP is a single-phase RNA extraction reagent containing guanidinium thiocyanate and phenol, which has been optimized to provide maximal recovery of the low-mass amounts of RNA in microparticles. Recovery of RNA is further improved by using the inert co-precipitant included in the kit. Cell-free fluids that can be processed with the ExoMir Kit include urine, blood serum, cerebrospinal fluid and eukaryotic cell culture media.
Recent research has highlighted the role of small membrane-bound particles such as exosomes and microvesicles, as intercellular mediators of biological information. Particles of various types and sizes are shed from solid tissues, leukocytes, and platelets, and find their way into the circulation and may also occur in other bodily fluids such as cerebrospinal fluid. Exosomes are among the smallest particles, with a size of ~ 30 – 100 nanometers. Exosomes originate when intracellular structures called multivesicular bodies fuse with the cell membrane and release their contents(exosomes) into the extracellular space. Multivesicular bodies are derived from the endosomal compartment, comprised of membrane-bound invaginations that can envelop and internalize exterior material. Microvesicles are larger particles and are shed directly from the cell membrane. Exosomes, microvesicles, and other membrane-bound micro particles are also shed into the culture media by eukaryotic cells propagated in vitro. Shed microparticles have been shown to transfer active mRNA and microRNA between cells. Release of microparticles may increase in number and type during pathological conditions, especially malignancies. Traditional methods for recovering exosomes involve centrifuging liquid samples at increasing centrifugal force, to sequentially pellet the larger and smaller particles.
The ExoMir™ PLUS kit is especially useful for allowing the initial sample processing steps to be carried out “in the field”, in situations where large-capacity centrifuges are not available. The ExoMir PLUS kit differs from the ExoMir Kit in that it has a third filter, which has a pore size of 700 nanometers (nm) and is used as the Top filter to trap the largest particles. Passing the sample through the 700 nm filter avoids the need to carry out an initial low-speed centrifugation of the sample to eliminate intact cells whose RNA content could obscure the RNA from the smaller particles. Including the 700 nm filter may also reduce clogging problems and allow higher sample volumes to be processed.
Selected Publications that Reference the ExoMir Kit:
Ali, S., et al. (2015) Contribution of microRNAs in understanding the pancreatic tumor microenvironment involving cancer associated stellate and fibroblast cells. Am J Cancer Res. 5(3): 1251–1264.
Bryant, R. J. et al. (2012) Changes in circulating microRNA levels associated with prostate cancer. British Journal of Cancer. doi:10.1038/bjc.2011.595.
Choi, H. Y., et al. (2014) Microparticles from kidney-derived mesenchymal stem cells act as carriers of proangiogenic signals and contribute to recovery from acute kidney injury. PLOS One. doi:10.1371/journal.pone.0087853.
de Candia, P., et al. (2013) Intracellular Modulation, Extracellular Disposal and Serum Increase of MiR-150 Mark Lymphocyte Activation. PLOS One. doi:10.1371/journal.pone.0075348.
Heishima, K., et al. (2015). MicroRNA-214 and MicroRNA-126 Are Potential Biomarkers for Malignant Endothelial Proliferative Diseases. International journal of molecular sciences, 16(10), 25377-25391.
Higashi, K., et al. (2015) MicroRNA-145 repairs infarcted myocardium by accelerating cardiomyocyte autophagy. American Journal of Physiology - Heart and Circulatory Physiology 309(11). H1813-H1826. doi: 10.1152/ajpheart.00709.2014.
Itoh, T. et al. (2012) Microvesicles released from hormone-refractory prostate cancer cells facilitate mouse pre-osteoblast differentiation. Journal of Molecular Histology. DOI: 10.1007/s10735-012-9415-1.
Le, M. T., Hamar, P., Guo, C., Basar, E., Perdigão-Henriques, R., Balaj, L., & Lieberman, J. (2014). miR-200–containing extracellular vesicles promote breast cancer cell metastasis. The Journal of clinical investigation, 124(12), 5109.
McGinn, C. (2014) The role of the haemodynamic Palladin protein within the Vasculature (Doctoral dissertation, School of Health and Human Performance, Dublin City University).
Minh, T. N. et. al. (2014) miR-200–containing extracellular vesicles promote breast cancer cell metastasis. J Clin Invest. doi:10.1172/JCI75695.
Pawlowski, T.L. et al. (2011) Comparison of miR expression patterns in plasma, serum, and cell line cMV. Cancer Res., 71: 4954.
Schageman J, Zeringer E , Li M, et al. (2013)The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo. BioMed Research International.
Takashima, Y. et al. (2014) Resetting Transcription Factor Control Circuitry toward Ground-State Pluripotency in Human. Cell 158(6): 1254-1269 doi: 10.1016/j.cell.2014.08.029.
Tsugita, M., et al. (2013) Ewing Sarcoma Cells Secrete EWS/Fli-1 Fusion mRNA via Microvesicles. PLOS One. doi:10.1371/journal.pone.0077416.
Vlassov, AV. et al. (2012) Exosomes: Current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. ScienceDirect, http://dx.doi.org/10.1016/j.bbagen.2012.03.017
Yamada, N. et al. (2014) Colorectal cancer cell-derived microvesicles containing microRNA-1246 promote angiogenesis by activating Smad 1/5/8 signaling elicited by PML down-regulation in endothelial cells. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms.doi: 10.1016/j.bbagrm.2014.09.002.
Yamada N, et al. (2013) Role of Intracellular and Extracellular MicroRNA-92a in Colorectal Cancer. Translational Oncology. 6(4): 482–492.
Yamada, N. and Akao, Y. (2016) Chapter Eight – Extracellular Vesicles in Cancer in Advances in Biomembranes and Lipid Self-Assembly. 23, 187–204. doi:10.1016/bs.abl.2016.01.004.
The ExoMir Kits contain sufficient components to treat and fractionate 10 samples of cell-free fluids and to extract RNA from both the filters as well as from the flow-through filtrate.
The shelf life of both of the ExoMir™ Kits is 12 months when stored properly.