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. A method for extracting DNA and RNA from a biological sample comprising:
(a) contacting the biological sample with a solid capture surface and a chemical crowding agent, thereby retaining cell-free DNA and 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;
(b) contacting the capture surface with a guanidine thiocyanate-based elution buffer while cell-free DNA and the microvesicles are on or in the capture surface, thereby releasing the DNA and RNA from the sample and producing a homogenate; and
(c) contacting the homogenate with a silica-based solid surface, thereby extracting the DNA, the RNA, or both the DNA and RNA from the homogenate.
2. The method of claim 1 , wherein the solid capture surface is magnetic.
3. The method of claim 1 , wherein the at least one anion exchange membrane or the at least one anion exchange bead is functionalized with quaternary ammonium moieties.
4. The method of claim 1 , 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.
5. The method of claim 1 , wherein the biological sample is plasma, serum, or urine.
6. The method of claim 1 , wherein the biological sample is between 0.2 to 20 mL.
7. The method of claim 1 , wherein the biological sample is urine.
8. The method of claim 7 , wherein the urine is first-catch urine.
9. The method of claim 1 , wherein the guanidine thiocyanate-based elution buffer comprises guanidine thiocyanate, a detergent, a buffer substance, or combinations thereof.
10. The method of claim 9 , wherein the guanidine thiocyanate-based elution buffer comprises guanidine thiocyanate and at least one of β-mercaptoethanol (BME), tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) a reducing agent.
11. The method of claim 1 , wherein step (c) further comprises adding protein precipitation buffer to the homogenate prior to extraction of the DNA, the RNA, or both the DNA and RNA from the homogenate, wherein the protein precipitation buffer has a defined pH range from 3.1 to 4.1.
12. The method of claim 11 , wherein the protein precipitation buffer further comprises a transition metal ion.
13. The method of claim 12 , wherein the transition metal ion is zinc.
14. The method of claim 1 , wherein the concentration of PEG is 0.5-10% (w/v).