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 .- 30 . (canceled)
31 . A method for extracting nucleic acids from a biological sample comprising a microvesicles comprising:
(a) contacting the biological sample with a capture surface under conditions sufficient to retain the microvesicles on or in the capture surface, wherein the capture surface comprises three membranes that are positively charged and functionalized with quaternary ammonium;
(b) lysing the microvesicles while the microvesicles are on or in the capture surface; and
(c) extracting the nucleic acids from the eluted microvesicles.
32 . The method of claim 31 , wherein the three membranes are identical to each other.
33 . The method of claim 31 , wherein the three membranes are directly adjacent to each other.
34 . The method of claim 31 , wherein the biological sample is plasma, serum, urine, cerebrospinal fluid or cell culture supernatant.
35 . The method of claim 31 , wherein the biological sample has a volume of less than about 4 mL.
36 . The method of claim 31 , wherein the biological sample has a volume of between about 0.1 to about 4 mL.
37 . The method of claim 31 , wherein the biological sample has a volume of about 1 mL.
38 . The method of claim 31 , wherein the extracted nucleic acids comprise mRNA, miRNA, or a combination thereof.
39 . A method for extracting nucleic acids from a biological sample comprising:
(a) contacting the biological sample with a capture surface under conditions sufficient to retain microvesicles on or in the capture surface, wherein the capture surface comprises three membranes that are positively charged and functionalized with quaternary ammonium;
(b) eluting the microvesicles from the capture surface to obtain a microvesicle fraction; and
(c) extracting the nucleic acids from the microvesicle fraction.
40 . The method of claim 39 , wherein the eluted microvesicle fraction from step (b) is concentrated by a spin concentrator to obtain a concentrated microvesicle fraction, and wherein the nucleic acids are extracted from the concentrated microvesicle fraction.
41 . The method of claim 39 , wherein the three membranes are identical to each other.
42 . The method of claim 39 , wherein the three membranes are directly adjacent to each other.
43 . The method of claim 39 , wherein the biological sample is plasma, serum, urine, cerebrospinal fluid or cell culture supernatant.
44 . The method of claim 39 , wherein the biological sample has a volume of less than about 4 mL.
45 . The method of claim 39 , wherein the biological sample has a volume of between about 0.1 to about 4 mL.
46 . The method of claim 39 , wherein the biological sample has a volume of about 1 mL.
47 . A kit comprising a:
a) a capture surface comprising three membranes that are positively charged and functionalized with quaternary ammonium;
b) at least one nucleic acid amplification primer, wherein the at least one nucleic acid amplification primer comprises at least one sequence selected from SEQ ID NOs: 1-10.
48 . The kit of claim 47 , wherein the at least one nucleic acid amplification primer comprises SEQ ID NO: 1.
49 . The kit of claim 47 , wherein the at least one nucleic acid amplification primer comprises SEQ ID NO: 2.
50 . The kit of claim 47 , further comprising a lysis buffer.