ELEMENT-TAGGED OLIGNUCLEOTIDE GENE EXPRESSION ANALYSIS

Abstract

Methods and kits for gene expression analysis are disclosed. The methods utilize element-tagged oligonucleotides as probes which are subsequently analyzed by elemental analysis. Also disclosed are methods and kits for the analysis of biological molecules using element labeled supports such as beads, followed by elemental analysis. The elemental analysis can be done using ICP-MS.

Description

BRIEF DESCRIPTION OF THE FIGURES

The skilled person in the art will understand that the figures, described below, are for illustration purposes only. The figures are not intended to limit the scope of the applicant's teaching in any way. The invention is illustrated in the figures, which are meant to be exemplary and not limiting.

FIG. 1(A) In situ hybridization and flow cytometry detection of 28S rRNA using biotinylated antisense oligonucleotdes (“oligos”) in three different conditions. (1)—corresponds to negative control cells hybridized with a nonsense biotinylated oligonucleotide (“oligo”), (2)—cells fixed with 4% para-formaldehyde 15 minutes, followed by Proteinase K (5 U/ml) for 15 minutes at room temperature and hybridized with 28S rRNA oligo; (3)—cells treated with 4% para-formaldehyde 15 minutes and Proteinase K (5 U/ml) for 15 minutes at 37° C. and hybridized with 28S rRNA oligo; (4)—cells fixed with 4% para-formaldehyde 15 minutes, followed by 0.3% Triton-X100, followed by Proteinase K (5 U/ml) for 15 minutes at 37° C. and hybridized with 28S rRNA oligo. Conditions denoted by (4) were chosen for further experiments. (B) Comparison of 28S rRNA in situ hybridization analyzed by flow cytometry (left graph) and ICP-MS (right graph).

FIG. 2. BCR/Abl (Break point cluster region/Abelson leukemia) gene expression analysis in leukemia cells by ICP-MS. (A) Schematic in situ hybridization of fixed/ permeabilized cells with a biotinylated oligonucleotide probe for BCR/Abl fusion gene. Biotin is identified by streptavidin (StrAv) labeled with terbium (Tb). Cell pellet is dissolved in HCL and analyzed by solution elemental ICP-MS analysis. (B) Experimental results for KG-1a cells (left graph) and K562 cells (right graph), hybridized with BCR/Abl antisense, 28S rRNA (positive control) and non-sense oligo probes (B/A) and no probe (ctrl); background and non-sense probe response values subtracted. Samples were run in triplicate. Data are presented as normalized ratio of terbium (Tb) to iridium (Ir) internal standard signal.

FIG. 3. Epidermal growth factor receptor (EGFR) gene expression analysis in adherent carcinoma cells by ICP-MS. A431 cells were hybridized with gene specific probes to EGFR, D-cyclin, 28S rRNA (positive control), and non-sense negative control, B/A. (B/A is a random oligo with random name used as negative control) Samples were run in triplicate. Data are presented as normalized ratio of terbium (Tb) to iridium (Ir) internal standard signal.

FIG. 4. Simultaneous protein and gene expression analysis in K562 leukemia cells by ICP-MS. (A) In situ hybridization with 28S rRNA and non-sense oligo probes (B/A); (B) immunolabeling of BCR/Abl protein and negative control IgG values during hybridization. Samples were run in triplicate. Data are presented as normalized ration of europium (Eu) or terbium (Tb) to iridium (Ir) internal standard signal.

FIG. 5. Work flow chart for in situ hybridization and gene expression analysis by ICP-MS.

FIG. 6. Work flow chart for element labeled bead gene expression analysis by particle elemental analysis.

FIG. 7. Work flow chart for simultaneous gene and protein expression analysis by ICP-MS.

Claims

1. A method for cellular analysis, comprising: (a) providing a cell or a cellular particle;(b) fixing and permeabilizing the cell or the cellular particle;(c) incubating the cell or the cellular particle in a hybridization solution with a probe specific for a target nucleic acid, the probe labeled with a unique element tag such that one type of said probe labeled with one type of said tag is distinguishable from any other type of said probe labeled with a different type of said tag by elemental analysis;(d) separating unhybridized probe from probe hybridized to the target nucleic acid by stringent washing conditions; and(e) analyzing the cell or cellular particle by elemental analysis to identify the probe and quantify the probe bound to the target nucleic acid.

2. The method of claim 1 wherein two or more differential probes labeled with differential element tags are hybridized to two or more target nucleic acids.

3. The method of claim 1 where the target nucleic acid is selected from the group consisting of intracellular nucleic acid molecules, matrix RNA, microRNA, gene transcript precursor RNA, messenger RNA, transport RNA, ribosomal RNA, chromosomal DNA, mitochondrial DNA, chloroplast DNA, viral DNA, viral RNA, bacterial DNA, bacterial RNA, and plasmid DNA.

4. The method of claim 1 further comprising simultaneous analysis of surface and/or intracellular protein molecules.

5. The method of claim 1 further comprising simultaneous analysis of surface and/or intracellular lipid molecules.

6. The method of claim 1 further comprising simultaneous analysis of surface and/or intracellular polysaccharide molecules.

7. The method of example 1 further comprising simultaneous analysis of surface and/or intracellular small molecules, selected from the group consisting of vitamins, hormones, haptens and nucleosides.

8. The method of claim 7 wherein the nucleosides are selected from the group consisting of ATP, ADP, cyclic AMP and NADH.

9. The method of claim 1 where after step (b) the cell or cellular particle is reacted with affinity reagents specific for surface or/and intracellular proteins, and the affinity reagents are labeled with element tags comprising a chemical moiety of a multitude of atoms of one or more isotopes of one or more elements attached to a supporting molecular structure, such that one type of said affinity reagent labeled with one type of said tag is distinguishable from any other type of said tag by elemental analysis, and followed by separating bound affinity reagents from unbound affinity reagents.

10. The method of claim 9 wherein the affinity reagents are selected from the group consisting of antibodies, aptamers, lectins and small molecules.

11. The method of claim 1 where after step (b) the cell or cellular particle is reacted with affinity reagents specific for surface or/and intracellular molecules and the affinity reagents are labeled with element tags, such that one type of said affinity reagent labeled with one type of said tag is distinguishable from any other type of said tag by elemental analysis, and followed by separating unbound affinity reagents from bound affinity reagents.

12. The method of claim 11 wherein the surface or/and intracellular molecules are lipids.

13. The method of claim 11 wherein the surface or/and intracellular molecules are polysaccharides.

14. The method of claim 11 wherein the surface or/and intracellular molecules are small molecules.

15. The method of claim 11 wherein the affinity reagents are selected from the group consisting of antibodies, aptamers, lectins and small molecules.

16. The method of claim 1 where the cell is a whole cell of an animal, plant, bacterium or fungus.

17. The method of claim 1 where the cellular particle is selected from the group consisting of an isolated chromosome, an isolated nucleus, an isolated mitochondria, an isolated chloroplast, an isolated virus, and an isolated bacterium.

18. The method of claim 1 where the probe is selected from the group consisting of an oligonucleotide probe, a locked nucleic acid (LNA) molecule, a peptide nucleic acid (PNA) molecule, a plasmid DNA, an amplified DNA or fragment thereof, an amplified RNA or fragment thereof, a fragment of RNA and a fragment of genomic DNA.

19. A method for homogeneous analysis of biological molecules in solution, comprising: (a) incubating biological molecules with affinity reagents labeled with element tags and uniquely tagged supports such that one type of said supports labeled with one type of said tags is distinguishable from any other type of said support labeled with a different type of said tags by elemental analysis, under conditions to enable the affinity reagents to bind with the biological molecules;(b) separating the supports with bound biological molecules from unbound supports; and(c) measuring the bound supports by particle elemental analysis wherein the supports are dispersed in a liquid to measure quantitatively the atomic and isotopic composition of individual supports, thereby detecting the types and the numbers of biological molecules attached to said supports.

20. The method of claim 19 wherein the supports are selected from the group consisting of particles, microspheres and beads.

21. The method of claim 19 wherein the biological molecules are from a tissue or a cell sample.

22. The method of claim 21 wherein the sample is selected from the group consisting of an animal sample, a plant sample, a bacterium sample, and a fungal sample.

23. The method of claim 19 wherein the biological molecules are selected from the group consisting of mRNA, protein, lipids, polysaccharides and small molecules.

24. The method of claim 19 where the binding of biological molecules with affinity reagents comprises the hybridization of mRNA molecules with oligonucleotides attached to uniquely tagged microspheres.

25. The method of claim 24 wherein the oligonucleotides comprise of a number of deoxythimidine triphosphate nucleosides and complementary nucleic acid probes attached to uniquely tagged microspheres.

26. The method of claim 25 wherein the complementary nucleic acid probes are selected from the group consisting of oligonucleotides, LNA, PNA and plasmid DNA.

27. The method of claim 19 further comprising the binding of the biological molecules to specific small molecules, wherein the small molecules are labeled with elemental tags which bind uniquely tagged supports coated with affinity reagents against the biological molecules, followed by elemental analysis to identify the reaction of said biological molecules with the small molecules.

28. The method of claim 27 wherein the affinity reagents are selected from the group consisting of antibodies, aptamers, and lectins, nucleic acids, binding peptides, protein receptors, phospholipids.

29. A kit for the detection and measurement of an element in a sample, where the measured element is an element tag attached to a specific probe complementary to a nucleic acid of interest, comprising: (a) an element tag for directly tagging a complementary probe; and(b) a complementary probe.

30. The kit of claim 29 further comprising instructions for i) direct tagging of the probe with the element tag; ii) fixing and permeabilizing a cell or cellular particle; iii) incubating the cell or cellular particle with the element tagged probe in a hybridization solution; iv) separating bound probe from unbound probe; v) dissolving the cell or cellular particle with hybridized material, and vi) detecting and measuring the element tagged probe

31. A kit for the detection and measurement of an element in a sample, where the measured element is an element tag attached to a specific probe complementary to a nucleic acid of interest, comprising: (a) a complementary probe tagged with an element tag.

32. The kit of claim 31 further comprising instructions for i) fixing and permeabilizing a cell or cellular particle; ii) incubating the cell or cellular particle with the element tagged probe in a hybridization solution; iii) separating bound probe from unbound probe; iv) dissolving the cell or cellular particle with hybridized material, and v) detecting and measuring the element tagged probe.

33. The kit of claim 30 wherein the detecting and measuring is done by solution elemental analysis.

34. The kit of claim 30 wherein the detecting and measuring is done by particle elemental analysis.

35. The kit of claim 29 further comprising a multitude of specific probes complementary to a multitude of nucleic acids and a multitude of unique element tags for uniquely labeling each type of probe.

36. The kit of claim 29 further comprising: (i) an affinity reagent for an intra or extracellular biological molecule selected from the group consisting of a protein, a lipid, a polysaccharide and a small molecule; and(ii) an elemental tag for labeling the affinity reagent for the biological molecule.

37. The kit of claim 36 further comprising instructions for (i) tagging the affinity reagent for the biological molecule, (ii) incubating the cell or cellular particle with the affinity reagent for the biological molecule; (iii) separating bound affinity reagent for the biological molecule from unbound reagent for the biological molecule; and (iv) detecting and measuring the bound reagent for the biological molecule.

38. The kit of claim 37 comprising a multitude of specific reagents for a multitude of biological molecules and a multitude of elemental tags for uniquely labeling each type of affinity reagent for each type of biological molecule.

39. A kit for the detection and measurement of an element, where the measured element is an element tag attached to oligo(dT)n and elements of uniquely labeled supports attached to complimentary probe, comprising: (a) an element tag for directly tagging oligo(dT)n;(b) oligo(dT)n;(c) at least one uniquely labeled support; and(d) a multitude of complementary probes.

40. The kit of claim 39 further comprising instructions for i) directly attaching the multitude of complementary probes to uniquely labeled supports; ii) performing nucleic acid purification; (iii) attaching the element tag to the oligo(dT)n; iv) hybridizing the complementary probes attached to uniquely labeled supports with purified nucleic acid; iii) reacting bound uniquely labeled supports with the metal tagged oligo(dT)n; iv) separating unbound nucleic acid from bound nucleic acids; v) detecting and measuring the elements of bound supports by elemental analysis.

41. The kit of claim 39 wherein there are a multitude of supports and the supports are particles or beads.

42. The kit of claim 39 wherein the multitude of complementary probes are directly tagged with distinguishable elemental tags.

43. A kit for the detection and measurement of an element, where the measured element is an element tag attached to oligo(dT)n and elements of uniquely labeled supports attached to complimentary probes, comprising: (a) an element tag for directly tagging oligo(dT)n;(b) oligo(dT)n; and(c) a multitude of complementary probes attached to at least one uniquely labeled supports.

44. The kit of claim 43 further comprising instructions for i) performing nucleic acid purification; (ii) attaching the element tag to the oligo(dT)n; iii) reacting the complementary probes with the element tagged oligo(dT)n; iv) hybridizing the complementary probes attached to uniquely labeled supports with purified nucleic acid; iii) reacting bound uniquely labeled supports with the metal tagged oligo(dT)n; iv) separating unbound nucleic acid from bound nucleic acid; v) detecting and measuring the elements of bound supports by elemental analysis.

45. A kit for the detection and measurement of an element, where the measured element is an element tag attached to oligo(dT)n and elements of uniquely labeled supports attached to complimentary probes, comprising: (a) an element tag labeled oligo(dT)n; and(b) a multitude of complementary probes attached to at least one uniquely labeled supports.

46. The kit of claim 45 further comprising instructions for i) performing nucleic acid purification; ii) hybridizing the complementary probes attached to uniquely labeled supports with purified nucleic acid; iii) reacting bound uniquely labeled supports with the metal tagged oligo(dT)n; iv) separating unbound nucleic acid from bound nucleic acid; v) detecting and measuring the elements of bound supports by elemental analysis.

47. The kit of claim 39 where the supports are beads.

48. The kit of claim 29 further comprising reagents and devices selected from the group consisting of dissociation solutions, spin columns with nucleic acid binding membranes, purification column for isolation and purification of nucleic acids from biological samples, reagents and solutions for amplification of purified nucleic acids, standards, dilution buffer, dissociation buffer, wash buffer, hybridization buffer and assay buffer.

49. The kit of claim 29 wherein endogenous nucleic acids are in situ amplified in morphologically intact cells.

50. The kit of claim 29 wherein the element is measured using a mass spectrometer.

51. The kit of claim 29 wherein the element is an isotope.

52. The kit of claim 29 wherein the element is selected from a group consisting of the noble metals, transition metals, rare earth elements, gold, silver, platinum, rhodium, iridium and palladium.

53. The kit of claim 29 wherein the element includes more than one element.

54. The kit of claim 29 wherein the element includes more than one isotope.

55. The kit of claim 29 wherein the element includes more than one atom of an isotope.

56. The kit of claim 36 wherein the affinity products are selected from the group consisting of antibody, Fab′, aptamer, antigen, hormone, growth factor, receptor, protein and nucleic acid.

57. The kit of claim 29 wherein instructions for particle elemental analysis is included.