Simultaneous quantification of gene expression in a user-defined region of a cross-sectioned tissue
The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein and/or nucleic acid expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.
1. A method comprising:
(1) contacting at least one nucleic acid target in a tissue sample with at least one probe comprising a target-binding domain, a signal oligonucleotide, and a photo-cleavable motif located between the target-binding domain and the signal oligonucleotide;
(2) illuminating a specific location of the tissue sample with light sufficient to release the signal oligonucleotide;
(3) collecting the released signal oligonucleotide by aspirating a solution proximal to the specific location that was illuminated, wherein the solution proximal to the specific location that was illuminated is located directly above the specific location that was illuminated, wherein the aspiration is performed using a capillary tube; and
(4) identifying the released signal oligonucleotide, thereby detecting the at least one nucleic acid target in the specific location of the tissue sample that was illuminated.
2. The method of claim 1 , wherein detecting comprises a polymerase reaction, a reverse transcriptase reaction, hybridization to an oligonucleotide microarray, mass spectrometry, hybridization to a fluorescent molecular beacon, a sequencing reaction, or molecular barcodes.
3. The method of claim 1 , wherein detecting comprises determining the identity and the abundance of the at least one nucleic acid target.
4. The method of claim 1 , wherein the at least one nucleic acid target comprises at least two distinct nucleic acid targets or at least two copies of the same nucleic acid target.
5. The method of claim 4 , wherein detecting comprises comparing the abundance of each distinct nucleic acid target.
6. The method of claim 1 , further comprising repeating at least steps (2)-(4) on at least a second specific location of the tissue sample.
7. The method of claim 6 , wherein detecting comprises comparing the abundance of the at least one nucleic acid target in the specific location and in the at least second specific location.
8. The method of claim 6 , wherein the specific location and the at least second specific location comprise the same cell type or distinct cell types.
9. The method of claim 1 , wherein the tissue sample is directly immobilized to a surface or is indirectly immobilized to a surface.
10. The method of claim 1 , wherein the tissue sample is a 2 to 1000 μm thick tissue section.
11. The method of claim 1 , wherein the tissue section is obtained from a formalin-fixed paraffin embedded (FFPE) sample.
12. The method of claim 1 , wherein the tissue sample is fixed or unfixed.
13. The method of claim 1 , wherein the tissue sample is stained or labeled thereby allowing visualization of a subcellular, cellular, or tissue-related structure in the stained or labeled tissue sample.
14. The method of claim 1 , wherein the signal oligonucleotide is a single-stranded nucleic acid or a partially double-stranded nucleic acid.
15. The method of claim 1 , wherein a negative purification is used to remove intact probe molecules from the released signal oligonucleotides, wherein the negative purification comprises an affinity purification comprising contacting an intact probe with an immobilized oligonucleotide that is complementary to a portion of the intact probe or an immobilized antibody or protein-binding motif that recognizes and binds to a portion of the intact probe, wherein the intact probe's target binding domain comprises a universal purification tag or sequence that is partially complementary to the immobilized oligonucleotide or is capable of being recognized or bound by the immobilized antibody or protein-binding motif.
16. The method of claim 1 , wherein the illumination is provided by a light source selected from the group consisting of an arc-lamp, a laser, a focused UV light source, and light emitting diode (LED).
17. The method of claim 1 , wherein the light is UV light.
18. The method of claim 1 , wherein the target-binding domain comprises a single-stranded nucleic acid or a partially double-stranded nucleic acid.
19. The method of claim 1 , wherein the capillary tube comprises an optical device capable of transmitting light to the specific location of the tissue sample.
20. The method of claim 1 , wherein the method further comprises illuminating a region of interest using a laser scanning device or a digital mirror device (DMD).