Methods for isolating a target nucleic acid from red blood cells
The invention provides a lysis reagent for lysing red blood cells, thereby releasing a target, such as RNA from a parasitic organism, in a form suitable for analysis. The reagent includes at least ammonium chloride and an anionic detergent, and may include an anti-coagulant. The reagent serves to lyse red blood cells, protect the released target from degradation in the lysate, and is compatible with subsequent steps for analysis of the target such as target capture, amplification, detection, or sequencing.
1. A method for isolating a target nucleic acid from red blood cells, the method comprising:
(a) mixing a sample containing red blood cells with a reagent comprising ammonium chloride at a concentration of 200-300 mM, lithium lauryl sulfate (LLS) at a concentration of 4% to 15% (w/v), and an anti-coagulant;
(b) incubating the mixture of step (a), wherein the red blood cells are lysed and a target nucleic acid in the red blood cells is released;
(c) adding a target capture oligomer and an immobilized probe to the mixture, wherein the target capture oligomer comprises (i) a target hybridizing sequence that hybridizes with the target nucleic acid and (ii) a segment for attaching the target capture oligomer to the immobilized probe, thereby forming a capture hybrid complex comprising the target capture oligomer and the target nucleic acid; and
(d) separating the capture hybrid complex away from other components of the sample, thereby isolating the target nucleic acid from the red blood cells.
2. The method of claim 1 , wherein the target nucleic acid is a pathogen-derived target.
3. The method of claim 1 , wherein the target nucleic acid is RNA.
4. The method of claim 2 , wherein the target nucleic acid is RNA.
5. The method of claim 1 , wherein the mixture further comprises a buffer.
6. The method of claim 5 , wherein the buffer is sodium bicarbonate.
7. The method of claim 6 , wherein the concentration of sodium bicarbonate is 5-30 mM.
8. The method of claim 7 , wherein the concentration of sodium bicarbonate is 10-20 mM.
9. The method of claim 6 , wherein the pH of the buffer is 7-8.
10. The method of claim 1 , wherein the anti-coagulant is EDTA or citrate.
11. The method of claim 1 , wherein the concentration of ammonium chloride in the reagent is 250 mM.
12. The method of claim 1 , wherein the concentration of LLS in the reagent is 5% to 8% (w/v).
13. The method of claim 12 , wherein the concentration of LLS in the reagent is 5% (w/v).
14. The method of claim 1 , wherein the concentration of ammonium chloride is 250 mM, the concentration of LLS is 5% (w/v), the concentration of sodium bicarbonate is 14 mM, and the pH is 7.2-7.6.
15. The method of claim 1 , wherein the segment for attaching the target capture oligomer to the immobilized probe is a poly-A sequence.
16. The method of claim 1 , wherein the immobilized probe is attached to a magnetic support.
17. The method of claim 1 , wherein the capture hybrid complex is separated away from other components of the sample using magnetic separation.
18. A method for isolating a target nucleic acid from red blood cells, the method comprising:
(a) mixing a sample containing red blood cells with a reagent comprising ammonium chloride at a concentration of 100-500 mM, lithium lauryl sulfate (LLS) at a concentration of 4% to 15% (w/v), sodium bicarbonate buffer at a concentration of 5-30 mM, and an anti-coagulant;
(b) incubating the mixture of step (a), wherein the red blood cells are lysed and a target nucleic acid in the red blood cells is released;
(c) adding a target capture oligomer and an immobilized probe to the mixture, wherein the target capture oligomer comprises (i) a target hybridizing sequence that hybridizes with the target nucleic acid and (ii) a segment for attaching the target capture oligomer to the immobilized probe, thereby forming a capture hybrid complex comprising the target capture oligomer and the target nucleic acid; and
(d) separating the capture hybrid complex away from other components of the sample, thereby isolating the target nucleic acid from the red blood cells.
19. The method of claim 18 , wherein the concentration of LLS in the reagent is 5% to 8% (w/v).
20. The method of claim 18 , wherein the capture hybrid complex is separated away from other components of the sample using magnetic separation.
21. A method for isolating a target nucleic acid from red blood cells, the method comprising:
(a) mixing a sample containing red blood cells with a reagent comprising ammonium chloride at a concentration of 100-500 mM, lithium lauryl sulfate (LLS) at a concentration of 4% to 15% (w/v), sodium bicarbonate buffer at pH 7-8, and an anti-coagulant;
(b) incubating the mixture of step (a), wherein the red blood cells are lysed and a target nucleic acid in the red blood cells is released;
(c) adding a target capture oligomer and an immobilized probe to the mixture, wherein the target capture oligomer comprises (i) a target hybridizing sequence that hybridizes with the target nucleic acid and (ii) a segment for attaching the target capture oligomer to the immobilized probe, thereby forming a capture hybrid complex comprising the target capture oligomer and the target nucleic acid; and
(d) separating the capture hybrid complex away from other components of the sample, thereby isolating the target nucleic acid from the red blood cells.
22. The method of claim 21 , wherein the concentration of LLS in the reagent is 5% to 8% (w/v).
23. The method of claim 21 , wherein the capture hybrid complex is separated away from other components of the sample using magnetic separation.