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 one or more beads that are positively charged and functionalized with quaternary ammonium;
(b) contacting the capture surface with a phenol-based lysis reagent 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;
(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 one or more beads are an anion exchanger functionalized with quaternary ammonium.
3. The method of claim 1 , wherein the biological sample is plasma or serum.
4. The method of claim 3 , wherein the biological sample is between 0.2 to 4 mL.
5. The method of claim 1 , wherein the biological sample is urine, cerebrospinal fluid or cell culture supernatant.
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 (b) further comprises a centrifugation step after contacting the capture surface with the phenol-based lysis reagent.
11. The method of claim 1 , wherein step (c) further comprises adding a control to the homogenate prior to extraction.
12. The method of claim 1 , wherein the method further comprises step (d) ethanol conditioning of the extraction from step (c); step (e) binding of the ethanol-conditioned extraction to a silica column; and step (f) eluting the extraction from the silica column.
13. The method of claim 1 , wherein the phenol-based lysis reagent comprises phenol and guanidinium.