Nucleic acid quantitation from tissue slides
View Patent ↗This invention provides methods of quantitating nucleic acids from problematic samples, such as aged samples, formalin fixed samples, paraffin embedded samples, samples with aneuploid cells, and cells with fragmented nucleic acids. Methods include techniques to efficiently solublize the nucleic acids under non-denaturing conditions from preserved clinical samples without resort to organic extractions, to normalize cell counts regardless of aneuploidy, to access the fragmentation state of the nucleic acids, and to provide standard curves for degraded nucleic acid samples.
1. A method of collecting a nucleic acid from formalin fixed paraffin embedded cells, said method comprising:
suspending the formalin fixed paraffin embedded cells in an aqueous solution;
incubating the suspension 3 hours or more at a temperature ranging from 40° C. to 100° C., and at least 2° C. below the Tm for double stranded DNA of the cells, whereby the paraffin melts and the nucleic acid is released from the cells into the aqueous solution, wherein the suspended cells incubated in the suspension are other than dewaxed cells; and,
physically separating the aqueous solution from the paraffin after said incubation, thereby collecting the nucleic acid from the cells;
wherein the paraffin has a melting point 71° C. or less and greater than 40° C.
2. The method of claim 1 , wherein said suspending comprises scraping the cells from a microscope slide, douncing or vortexing.
3. The method of claim 1 , wherein the aqueous solution comprises constituents selected from the group consisting of: PEG (polyethylene glycol), SDS (sodium dodecyl sulfate), SSC (saline-sodium citrate) buffer, NaHPO 4 , EDTA (ethylene diamine tetra-acetate), denatured salmon sperm DNA (deoxyriboneucleic acid), formamide, and SSPE (saline-sodium phosphate EDTA) buffer.
4. The method of claim 1 , wherein the aqueous solution comprises constituents selected from the group consisting of: blocking probes, capture extenders, label extenders, preamplifiers, label probes, amplification probes, amplification multimers, a protease, a lipase, a surfactant, or nuclease inhibitor.
5. The method of claim 1 , wherein the aqueous solution comprises more than 150 ug/ml of a proteinase.
6. The method of claim 5 , wherein the protease comprises a proteinase K.
7. The method of claim 1 , wherein the incubation temperature comprises a temperature ranging from 52° C. to less than 80° C.
8. The method of claim 1 , wherein the incubation temperature comprises a temperature of 65° C.
9. The method of claim 1 , wherein said incubating comprises holding the suspension at the incubation temperature for 12 hours or more.
10. The method of claim 1 , wherein said non-denaturing conditions include pH conditions that do not exceed pH 8.5 or a temperature of 70° C.
11. The method of claim 1 , wherein said separating comprises a technique selected from the group consisting of: centrifuging, decanting, aspirating, filtering, pipetting, and solidifying the hydrophobic component at a temperature below the melting point.
12. The method of claim 1 , further comprising capturing the nucleic acid on a solid support.
13. The method of claim 1 , further comprising quantitating the nucleic acid by detecting the nucleic acid and comparing the detected nucleic acid to a standard.
14. The method of claim 13 , wherein said detecting comprises a technique selected from the group consisting of: bDNA analysis, northern blot analysis, Southern blot analysis, polymerase chain reaction, nucleic acid sequencing techniques, and agarose gel electrophoresis.
15. The method of claim 1 , further comprising phenol extraction of the separated aqueous solution.
16. The method of claim 1 , wherein the aqueous solution is not chemically extracted with an organic solvent.
17. The method of claim 1 , wherein the incubation temperature is at least 2° C. above the melting point temperature of the hydrophobic component.
18. The method of claim 1 , wherein the incubation temperature ranges from 45° C. to 95° C.
19. The method of claim 1 , wherein the incubation temperature is at least 5° C., or 10° C. below a Tm of the cell DNA.
20. The method of claim 1 , wherein the incubation temperature is less than a temperature that would denature a preponderance of the cell DNA.