Methods for isolating microvesicles and extracting nucleic acids from biological samples
The invention provides novel methods and kits for isolating nucleic acids from biological samples, including cell-free DNA and/or cell-free DNA and nucleic acids including at least RNA from microvesicles, and for extracting nucleic acids from the microvesicles and/or from the biological samples.
1. A method for extracting cell-free DNA and microvesicular RNA from a biological sample comprising:
(a) contacting the biological sample with a capture surface under conditions sufficient to retain cell-free DNA and microvesicles from the biological sample on or in the capture surface, wherein the capture surface comprises a membrane that is positively charged and functionalized with quaternary ammonium;
(b) contacting the capture surface with a guanidinium thiocyanate-based extraction buffer while cell-free DNA and the microvesicles are on or in the capture surface, thereby releasing the cell-free DNA and microvesicular RNA from the sample and producing a homogenate; and
(c) extracting the cell-free DNA, the microvesicular RNA, or both the cell-free DNA and microvesicular RNA from the homogenate.
2. The method of claim 1 , wherein the membrane is an anion exchanger functionalized with quaternary ammonium.
3. The method of claim 1 , wherein the capture surface comprises three membranes that are positively charged and functionalized with quaternary ammonium.
4. The method of claim 1 , wherein the biological sample is plasma, serum, urine, cerebrospinal fluid or cell culture supernatant.
5. The method of claim 1 , wherein the biological sample is between 0.2 to 4 mL.
6. The method of claim 1 , wherein step (a) further comprises processing the biological sample by filtering the biological sample.
7. The method of claim 6 , wherein the filtration is performed using a 0.8 μm filter.
8. The method of claim 1 , wherein step (a) further comprises a centrifugation step after contacting the biological sample with the capture surface.
9. The method of claim 1 , wherein step (a) further comprises washing the capture surface after contacting the biological sample with the capture surface.
10. The method of claim 1 , wherein step (c) further comprises a centrifugation step after contacting the capture surface with the guanidinium thiocyanate-based extraction buffer.
11. A method for extracting cell-free DNA and microvesicular RNA from a biological sample comprising:
(a) contacting the biological sample with a capture surface under conditions sufficient to retain cell-free DNA and microvesicles from the biological sample on or in the capture surface, wherein the capture surface comprises one or more beads that are positively charged and functionalized with quaternary ammonium;
(b) contacting the capture surface with a guanidinium thiocyanate-based extraction buffer while cell-free DNA and the microvesicles are on or in the capture surface, thereby releasing the cell-free DNA and microvesicular RNA from the sample and producing a homogenate; and
(c) extracting the cell-free DNA, the microvesicular RNA, or both the cell-free DNA and microvesicular RNA from the homogenate.
12. The method of claim 11 , wherein the one or more beads are an anion exchanger functionalized with quaternary ammonium.
13. The method of claim 11 , wherein the biological sample is plasma, serum, urine, cerebrospinal fluid or cell culture supernatant.
14. The method of claim 11 , wherein the biological sample is between 0.2 to 4 mL.
15. The method of claim 11 , wherein step (a) further comprises processing the biological sample by filtering the biological sample.
16. The method of claim 15 , wherein the filtration is performed using a 0.8 μm filter.
17. The method of claim 11 , wherein step (a) further comprises a centrifugation step after contacting the biological sample with the capture surface.
18. The method of claim 11 , wherein step (a) further comprises washing the capture surface after contacting the biological sample with the capture surface.
19. The method of claim 11 , wherein step (b) further comprises a centrifugation step after contacting the capture surface with the guanidinium thiocyanate-based extraction buffer.