# US 10,767,214 B2 · Method of microvesicle enrichment > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,767,214 B2 - Document ID: US10767214B2 - Status: Granted - Kind: B2 - Application number: 14/354,111 - Filed: 2011-10-26 - Published: 2015-01-08 - Granted: 2020-09-08 - Earliest claimed: 2011-10-26 - Assignees: Clinical Genomics Pty. Ltd. - Applicants: Lawrence Charles LaPointe; Susanne Kartin Pedersen; Aidan McEvoy - Inventors: Lawrence Charles LaPointe; Susanne Kartin Pedersen; Aidan McEvoy - Web page: https://patentgrep.com/patent/US10767214B2 ## CPC - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract The present invention relates to a method of enriching for membranous microvesicles relative to the cellular population in a biological sample. More particularly, there is provided a method for enriching for exosomes from plasma. In a related aspect, there is provided a method of reducing the concentration of cellular and cellular derived molecules in a biological sample. Still further, the present invention provides methods for selectively isolating mRNA subpopulations from exosomes. Yet further, there are provided methods of amplifying exosome derived RNA. The method of the present invention is useful in a range of applications including, but not limited to, diagnostic, prognostic, therapeutic, research and development applications, to the extent that the enrichment of exosomes is required. ## Claims ### Claim 1 A method of enriching for membranous microvesicles from a mammalian biological sample, the method comprising (a) subjecting the biological sample to a single step membranous microvesicle enrichment, wherein the single step consists of mechanical cellular rupture selected from the group consisting of (i) centrifugation; (ii) sonication, with or without the inclusion of surfactants; (iii) bead milling with or without the addition of surfactants using small glass; ceramic, zirconium or steel beads; (iv) homogenization; (v) using a nitrogen burst method; (vi) using a small probe ultrasound; (vii) using hypotonic shock; (viii) using high-shear mechanical methods; (ix) using rotor-stator disruptors; (x) using valve-type processors; (xi) using fixed geometry processors; (xii) using constant pressure processors; (xiii) using osmosis based electroporation; and (xiv) electropermeabilization, and wherein the mechanical cellular rupture selectively ruptures residual cells but not membranous microvesicles, and (b) lysing the enriched membranous microvesicles resulting from the single step in (a) to release their nucleic acid and protein content. ### Claim 2 (depends on 1) The method of claim 1, wherein the membranous microvesicles are exosomes. ### Claim 3 (depends on 1) The method of claim 1, wherein the mechanical cell rupture is performed using centrifugation at 15,000-17,000 g or bead milling. ### Claim 4 (depends on 3) The method of claim 3, wherein the bead is about 0.1 mm-0.5 mm in diameter and the sample is milled at about 20-40 Hz. ### Claim 5 (depends on 4) The method of claim 4, wherein the bead is a 0.1 mm glass bead and the milling is performed at about 30 Hz for about 2-5 minutes. ### Claim 6 (depends on 1) The method of claim 1, wherein the nucleic acid is mRNA and the method further comprises: isolating polyadenylated RNA. ### Claim 7 (depends on 6) The method of claim 6, wherein the polyadenylated RNA is isolated using oligo(dT) affinity chromatography; and wherein the method further comprises amplifying the isolated polyadenylated RNA. ### Claim 8 (depends on 7) The method of claim 7, wherein the amplification is selected from the group consisting of 1-step PCR and 2-step PCR. ### Claim 9 (depends on 8) The method of claim 8, wherein the amplification is 1-step PCR. ### Claim 10 (depends on 9) The method of claim 9, wherein the bead is about 0.1 mm-0.5 mm in diameter and the sample is milled at about 20-40 Hz or about 30 Hz. ### Claim 11 (depends on 10) The method of claim 10, wherein the bead is a 0.1 mm glass bead and the milling is performed at about 30 Hz for about 2-5 minutes. ### Claim 12 (depends on 9) The method of claim 9, wherein the mRNA has been eluted in about 35 μl of RNase free water and is amplified using 1-step PCR in a reaction volume of about 50 μl. ### Claim 13 (depends on 7) The method of claim 7, wherein isolation of the polyadenylated RNA comprises binding to a silica column, washing with Buffer ACWI (50-100% Guanidium chloride, Ethanol), Buffer ACW2 (50-100% Guanidium chloride, Ethanol) and ethanol and eluting in Buffer AVE (RNase-free water with 0.04% sodium-azide (v/v) and wherein: (a) the ACW2 buffer wash step is performed twice; (b) the ethanol wash step is performed twice; (c) the RNA is eluted in a final volume of about 35 μl; and (d) the eluate of step (c) and Buffer AVE are reapplied to the column. ### Claim 14 (depends on 1) The method of claim 1, wherein the biological sample is blood, serum, plasma, urine, stool, ascites, cerebrospinal fluid or saliva. ### Claim 15 (depends on 1) The method of claim 1, further comprising contacting an enzyme with the biological sample prior to or during the mechanical cellular rupture step (a). ### Claim 16 (depends on 15) The method of claim 15, wherein the enzyme is a ribonuclease or a deoxyribonuclease. ### Claim 17 A method of isolating RNA from membranous microvesicles, comprising: selectively rupturing cells in a plasma sample to release cellular nucleic acid content; degrading the cellular nucleic acid content with an enzyme; neutralizing the enzyme; enriching membranous microvesicles, wherein the membranous microvesicles comprise RNA; and lysing the membranous microvesicles to release RNA. ### Claim 18 (depends on 17) The method of claim 17, wherein selective rupturing comprises centrifugation or bead milling. ### Claim 19 (depends on 17) The method of claim 17, wherein the enzyme is ribonuclease. ### Claim 20 (depends on 17) The method of claim 17, wherein the RNA is polyadenylated RNA.