Nucleic acid extraction from heterogeneous biological materials
Methods for extracting high quality nucleic acids from a heterogenous collection of nucleic acid-containing materials from a biological sample are disclosed. The heterogenous collection of nucleic-acid containing materials may contain cells or microvesicles, or both. The extractions obtained by the methods described herein are characterized by high yield and high integrity, making the extracted nucleic acids useful for various applications in which high quality nucleic acid extractions are preferred, e.g., a diagnosis, prognosis, or therapy evaluation for a medical condition.
1. A method of extracting nucleic acid from a human biological sample, wherein the human biological sample comprises a heterogeneous collection of microvesicles, the method comprising the steps of:
a) filtering or centrifuging the biological sample to obtain a fraction comprising the heterogeneous collection of microvesicles, wherein the filtration or centrifugation excludes lipids, debris from dead cells, contaminants and PCR inhibitors;
b) depleting the fraction of at least 50% of microvesicles derived from erythrocytes by contacting the fraction with an affinity agent that binds to at least one surface marker to remove microvesicles that comprise the at least one surface marker from the fraction, wherein the surface marker is glycophorin A (CD235);
c) contacting the fraction from step (b) with at least one RNase inhibitor at least one reducing agent, at least one decoy substrate, at least one soluble receptor, at least one small interfering RNA, at least one RNA binding molecule, at least one RNase denaturing substance, or any combination thereof;
d) performing a processing step on the fraction from step (c), comprising washing the microvesicles, removing any RNases by size separation, denaturing any proteins, or a combination thereof;
e) extracting nucleic acid from the fraction from step (d).
2. The method of claim 1 , further comprising contacting the fraction with an affinity agent that binds to at least one second surface marker selected from the markers listed in Table 1 or Table 2 to enrich and purify nucleic acid-containing materials that comprise the at least one second surface marker.
3. The method of claim 2 , wherein the at least one second surface marker is selected from P-selectin, CD45, L1 cam, CD44, CD184, PDGFR, RH, CD3, CD19, CD20, CD56, CD11, CD14, CD90, CD326, or CD324.
4. The method of claim 3 , wherein the at least one second surface marker is selected from L1cam, CD45, CD3, CD44 or CD184.
5. The method of claim 1 , wherein the human biological sample is a bodily fluid.
6. The method of claim 5 , wherein the bodily fluid is urine.
7. The method of claim 5 , wherein the bodily fluid is serum or plasma.
8. The method of claim 1 , wherein the RNase inhibitor is a protease.
9. The method of claim 1 , wherein the nucleic acid obtained from practice of the extraction method is analyzed for the presence or absence of a genetic aberration associated with a disease or medical condition.
10. The method of claim 1 , wherein the decoy substrate comprises synthetic RNA; or wherein the RNA binding molecule comprises an anti-RNA antibody, chaperone protein, or an RNase inhibitory protein; or wherein the RNase denaturing substance comprises a high osmolarity solution or detergent.
11. The method of claim 1 , wherein the affinity agent comprises an antibody, an aptamer, an aptamer analog or a molecularly imprinted polymer specific for the marker.
12. The method of claim 1 , wherein steps b and c occur sequentially.
13. The method of claim 1 , wherein steps b and c occur concurrently.
14. The method of claim 1 , wherein steps c and d occur sequentially.
15. The method of claim 1 , wherein steps c and d occur concurrently.