US 10,767,214 B2Granted

Method of microvesicle enrichment

AssigneeClinical Genomics Pty. Ltd.

InventorsLawrence Charles LaPointe, Susanne Kartin Pedersen, Aidan McEvoy

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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.

Claims20

Dependent claims

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.

16 more elements

Dependencies are inferred from the claim text.