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.
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.
de Candia P, Torri A, Gorletta T, Fedeli M, Bulgheroni E, et al. (2013) Intracellular Modulation, Extracellular Disposal and Serum Increase of MiR-150 Mark Lymphocyte Activation. PLoS ONE 8(9): e75348. doi:10.1371/journal.pone.0075348
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
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.
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, Nakagawa Y, et al. (2013) Role of Intracellular and Extracellular MicroRNA-92a in Colorectal Cancer. Translational Oncology. 6(4): 482–492.
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.