METHODS FOR ISOLATING MICROVESICLES
The invention provides novel methods for isolating microvesicles from a biological sample and for extracting nucleic acids from the microvesicles.
1 . A method for extracting nucleic acids from a biological sample comprising:
(a) providing a biological sample;
(b) contacting the biological sample with a capture surface under conditions sufficient to retain the microvesicle fraction on or in the capture surface;
(c) lysing the microvesicle fraction while the microvesicles are on or in the capture surface; and
(d) extracting the nucleic acids from the microvesicle fraction.
2 . The method of claim 1 , wherein the capture surface is positively charged.
3 . The method of claim 1 , wherein the capture surface is a membrane.
4 . The method of claim 3 , wherein the membrane comprises regenerated cellulose.
5 . The method of claim 3 , wherein the membrane has a pore size of at least 3 um.
6 . The method of claim 3 , wherein the membrane is functionalized with quaternary ammonium R—CH 2 —N + (CH 3 ) 3 .
7 . The method of claim 1 , wherein the capture surface comprises more than one membrane.
8 . The method of claim 7 , wherein at least one membrane is charged.
9 . The method of claim 7 , wherein the capture surface comprises at least two membranes, where each membrane has a different charge.
10 . The method of claim 7 , wherein the capture surface comprises three membranes, wherein said three membranes are directly adjacent to one another.
11 . The method of claim 10 , wherein the three membranes are identical to each other.
12 . The method of claim 1 , wherein the biological sample is plasma, serum, urine, cerebrospinal fluid or cell culture supernatant.
13 . The method of claim 1 , wherein step (a) further comprises contacting the biological sample with a loading buffer.
14 . The method of claim 13 , wherein the loading buffer has a neutral pH.
15 . The method of claim 1 , wherein the nucleic acids comprise RNA.
16 . The method of claim 15 , wherein the RNA comprises mRNA, miRNA, or a combination thereof.
17 . The method of claim 1 , wherein the nucleic acids comprise DNA.
18 . A method for extracting nucleic acids from a biological sample comprising:
(a) providing a biological sample;
(b) contacting the biological sample with a capture surface under conditions sufficient to retain the microvesicle fraction on or in the capture surface;
(c) eluting the microvesicles from the capture surface to obtain a microvesicle fraction; and
(d) extracting the nucleic acids from the microvesicle fraction.
19 . The method of claim 18 , wherein the eluted microvesicle fraction from step (c) is concentrated by a spin concentrator to obtain a concentrated microvesicle fraction, and wherein the nucleic acids are extracted from the concentrated microvesicle fraction.
20 . The method of claim 18 , wherein the capture surface is positively charged.
21 . The method of claim 18 , wherein the capture surface is a membrane.
22 . The method of claim 21 , wherein the membrane comprises regenerated cellulose.
23 . The method of claim 21 , wherein the membrane has a pore size of at least 3 um.
24 . The method of claim 21 , wherein the membrane is functionalized with quaternary ammonium R—CH 2 —N + (CH 3 ) 3 .
25 . The method of claim 18 , wherein the capture surface comprises more than one membrane.
26 . The method of claim 25 , wherein at least one membrane is charged.
27 . The method of claim 25 , wherein the capture surface comprises at least two membranes, where each membrane has a different charge.
28 . The method of claim 25 , wherein the capture surface comprises three membranes, wherein said three membranes are directly adjacent to one another.
29 . The method of claim 28 , wherein the three membranes are identical to each other.
30 . The method of claim 18 , wherein the biological sample is plasma, serum, urine, cerebrospinal fluid or cell culture supernatant.