AUTOMATED AND MANUAL METHODS FOR ISOLATION OF EXTRACELLULAR VESICLES AND CO-ISOLATION OF CELL-FREE DNA FROM BIOFLUIDS
The invention provides novel methods and kits for fully automated high-throughput method for isolation of extracellular vesicles and co-isolation of cell-free DNA from biofluids, including cell-free DNA and/or cell-free DNA and nucleic acids including at least RNA from microvesicles, novel methods and kits for isolation of extracellular vesicles and co-isolation of cell-free DNA from biofluids, including cell-free DNA and/or cell-free DNA and nucleic acids including at least RNA from microvesicles that do not require the use of phenol or chloroform, and for extracting nucleic acids from the extracellular vesicles and/or from the biological samples.
1 - 100 . (canceled)
101 . A method for isolating microvesicles from a biological sample comprising:
(a) contacting the biological sample with a solid capture surface and a chemical crowding agent, thereby retaining microvesicles from the biological sample on or in the capture surface, wherein the chemical crowding agent comprises polyethylene glycol, wherein the solid capture surface comprises at least one anion exchange membrane or at least one anion exchange bead; and
(b) eluting the microvesicles from the solid capture surface, thereby isolating the microvesicles from the biological sample.
102 . The method of claim 101 , wherein the microvesicles are eluted from the solid capture surface in step (b) using a change ionic strength.
103 . The method of claim 101 , wherein the microvesicles are eluted from the solid capture surface in step (b) using a change in pH.
104 . The method of claim 101 , further comprising:
(c) lysing the microvesicles eluted from the solid capture surface in step (b), thereby producing a homogenate; and
(d) extracting nucleic acids from the homogenate.
105 . The method of claim 104 , wherein step (c) comprises contacting the microvesicles with a guanidine thiocyanate-based lysis reagent.
106 . The method of claim 104 , wherein step (d) comprises contacting the homogenate with a silica-based solid surface.
107 . The method of claim 101 , wherein the solid capture surface is magnetic.
108 . The method of claim 101 , wherein the at least one anion exchange membrane or the at least one anion exchange bead is functionalized with quaternary ammonium moieties.
109 . The method of claim 101 , wherein the at least one anion exchange membrane or the at least one anion exchange bead is functionalized with sulfate, sulfonate, tertiary amine, any other IEX group, or any combination thereof.
110 . The method of claim 101 , wherein the biological sample is plasma, serum, or urine.
111 . The method of claim 101 , wherein the biological sample is between 0.2 to 20 mL.
112 . The method of claim 101 , wherein the biological sample is urine.
113 . The method of claim 112 , wherein the urine is first-catch urine.
114 . The method of claim 105 , wherein the guanidine thiocyanate-based lysis reagent comprises guanidine thiocyanate and at least one of a detergent and a buffer substance.
115 . The method of claim 105 , wherein the guanidine thiocyanate-based lysis reagent comprises guanidine thiocyanate and at least one of β-mercaptoethanol (BME), tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT).
116 . The method of claim 104 , wherein step (d) further comprises adding protein precipitation buffer to the homogenate prior to extraction of the nucleic acids from the homogenate, wherein the protein precipitation buffer has a defined pH range from 3.1 to 4.1.
117 . The method of claim 116 , wherein the protein precipitation buffer further comprises a transition metal ion.
118 . The method of claim 117 , wherein the transition metal ion is zinc.
119 . The method of claim 101 , wherein the concentration of PEG is 0.5-10% (w/v).