Methods and product for optimising localised or spatial detection of gene expression in a tissue sample

Abstract

The present invention relates to methods and products for localized or spatial detection and/or analysis of RNA in a tissue sample or a portion thereof, comprising: (a) providing an object substrate on which at least one species of capture probe, comprising a capture domain, is directly or indirectly immobilized such that the probes are oriented to have a free 3′ end to enable said probe to function as a reverse transcriptase (RT) primer; (b) contacting said substrate with a tissue sample and allowing RNA of the tissue sample to hybridise to the capture probes; (c) generating cDNA molecules from the captured RNA molecules using said capture probes as RT primers; (d) labelling the cDNA molecules generated in step (c), wherein said labelling step may be contemporaneous with, or subsequent to, said generating step; (e) detecting a signal from the labelled cDNA molecules; and optionally (f) imaging the tissue sample, wherein the tissue sample is imaged before or after step (c).

The invention claimed is: 
     
       1. A method for localized or spatial detection and/or analysis of RNA in a tissue sample or a portion thereof section, comprising:
 (a) providing an object substrate on which at least one species of capture probe, comprising a capture domain, is directly or indirectly immobilized on the object substrate such that the probes are capture probe is oriented to have a free 3′ end to enable said capture probe to function as a reverse transcriptase (RT) primer, 
 (b) contacting said object substrate with a tissue sample section and allowing RNA of the tissue sample section to directly hybridise to the capture probes under a set of conditions; 
 (c) generating first strand cDNA molecules from the captured hybridised RNA molecules using said capture probes as RT primers and a fluorescently labelled nucleotide, thereby incorporating the fluorescently labelled nucleotide into the cDNA molecules, 
 wherein said incorporation of the fluorescently labelled nucleotide is contemporaneous with said generating step; 
 (d) labelling the cDNA molecules generated in step (c), wherein said labelling step may be contemporaneous with, or subsequent to, said generating step; 
 (e) (d) detecting a signal from the fluorescently labelled cDNA molecules on the object substrate, thereby spatially detecting RNA of the tissue section; and optionally 
 (f)(e) imaging the tissue samplesection, wherein the tissue samplesection is imaged before or after step (c), and combining the signal from the fluorescently labelled cDNA molecules with the image of the tissue section, thereby spatially detecting the RNA in the tissue section. 
 
     
     
       2. The method of  claim 1 , being a method for determining the optimum conditions for localised or spatial detection of RNA in a tissue sample section on an object substrate, comprising:
 (g)(f) repeating steps (a)-(e), and optionally step (f), using a second set of conditions that are different to the conditions used in step (b); 
 (h)(g) comparing thesignal intensity and/or resolution of the signal from the labelled cDNA molecules immobilized on the object substrate; and optionally 
 (i)(h) selecting, from the conditions used in step (b) and the second set of conditions used in step (g)(f), the conditions that provide the optimum signal intensity and/or resolution of the labelled cDNA molecules. 
 
     
     
       3. The method of  claim 1 , being a method for determining and/or analysing RNA or a transcriptome of a tissue sample or a portion thereof section comprising further steps:
 (g′) (f′) removing the labelled cDNA from at least one portion of the surface of the object substrate; 
 (h′) (g′) optionally amplifying the remaining cDNA molecules immobilized on the surface of the object substrate; 
 (i′) (h′) releasing at least part of the remaining cDNA molecules and/or optionally their amplicons from the surface of the object substrate, wherein said released molecules may be a first strand and/or second strand cDNA molecule or an amplicon thereof; and  
 (j′) (i′) directly or indirectly analysing the sequences of the released cDNA molecules or the amplicon thereof. 
 
     
     
       4. The method of  claim 3 , further comprising step (k) (j) correlating said sequence analysis information the released cDNA molecule or amplicon sequences with an image of said tissue sample section, wherein the tissue sample section is imaged before or after step (c). 
     
     
       5. The method of  claim 3 , wherein the at least one portion of labelled cDNA molecules is removed from from the at least one portion of the surface of the object substrate is removed by laser ablation. 
     
     
       6. The method of  claim 1 , being a method for determining and/or analysing RNA or a transcriptome of a tissue sample or a portion thereof section comprising:
 (a″) providing an object substrate on which multiple species of capture probes are directly or indirectly immobilized such that each species of capture probe occupies a distinct position on the object substrate and is oriented to have a free 3′ end to enable said capture probe to function as a reverse transcriptase (RT) primer, wherein each species of said capture probe comprises a nucleic acid molecule with 5′ to 3′: 
 (i) a positional domain that corresponds to the position of the capture probe on the object substrate, and 
 (ii) a capture domain; 
 (b″) contacting said object substrate with a tissue sample section such that the position of a the capture probe probes on the object substrate may be correlated with a position in the tissue sample section and allowing RNA of the tissue sample section to hybridise to the capture domain in said capture probes under a set of conditions; 
 (c″) generating cDNA molecules from the captured hybridised RNA molecules using said capture probes as RT primers, 
 (d″) labelling the cDNA molecules generated in step (c′), wherein said labelling step may be is contemporaneous with , or subsequent to, said generating step; 
 (e″) detecting a signal from the labelled cDNA molecules; 
 (f″) optionally imaging the tissue sample section, wherein the tissue sample section is imaged before or after step (c″); 
 (g″) optionally removing the labelled cDNA molecules from at least one portion of the surface of the object substrate; 
 (h″) optionally amplifying the remaining cDNA molecules immobilized on the surface of the object substrate; 
 (i″) releasing at least part of the remaining cDNA molecules and/or optionally their amplicons from the surface of the object substrate, wherein said released molecules may be a first strand and/or second strand cDNA molecule or an amplicon thereof and wherein said part includes the positional domain or a complement thereof; and  
 (j″) directly or indirectly analysing the sequence sequences of the released cDNA molecules or the amplicons thereof. 
 
     
     
       7. The method of  claim 6 , wherein the remaining cDNA molecules are released from the surface of the object substrate by:
 (i) nucleic acid cleavage; 
 (ii) denaturation; and/or 
 (iii) physical means. 
 
     
     
       8. The method of  claim 7 , wherein the remaining cDNA molecules are released by enzymatic cleavage of a cleavage domain, which is located in the a universal domain or positional domain of the capture probe; or wherein the remaining cDNA molecules are released by applying hot water or buffer to the object substrate. 
     
     
       9. The method of  claim 6 , further comprising a step of washing the object substrate to remove residual tissue section. 
     
     
       10. The method of  claim 6 , wherein each species of capture probe is immobilized on the object substrate by bridge amplification to form a local clonal colony of capture probe such that each species of capture probe occupies a distinct position on the object substrate. 
     
     
       11. The method of  claim 6 , wherein the object substrate is a bead array. 
     
     
       12. The method of  claim 11 , wherein each species of capture probe is immobilized on a different bead such that each species of capture probe occupies a distinct position on the object substrate. 
     
     
       13. The method of  claim 1 , further comprising a step of correlating the signal detected from the labelled cDNA molecules with an image of said tissue sample, wherein the tissue sample is imaged before or after step (c). 
     
     
       14. The method of  claim 1 , wherein the label is incorporated into the cDNA molecules generated in step (c). 
     
     
       15. The method of  claim 14 , wherein the label is conjugated to a nucleotide and the step of labelling comprises the incorporation of labelled nucleotides into the synthesized cDNA molecule. 
     
     
       16. The method of  claim 15 , wherein the lapelled nucleotides are fluorescently labelled nucleotides. 
     
     
       17. The method of  claim 1 , wherein the step of detecting a signal from the labelled cDNA molecules comprises imaging the object substrate such that the signal from the labelled cDNA molecules is detected. 
     
     
       18. The method of  claim 17 , being a method for the identification of transcriptionally active tumour cells, wherein the tissue sample is a cell suspension comprising section comprises tumour cells and the image of the labelled cDNA on the object substrate corresponds to transcriptionally active cells. 
     
     
       19. The method of  claim 17 , wherein the object substrate is imaged using light, bright field, dark field, phase contrast, fluorescence, reflection, interference or confocal microscopy or a combination thereof. 
     
     
       20. The method of  claim 1 , wherein the capture probes are DNA molecules. 
     
     
       21. The method of  claim 1 , wherein the capture probes further comprise a positional domain which is 5′ relative to the capture domain, wherein said positional domain comprises a sequence that corresponds to the position of the capture probe position on the object substrate. 
     
     
       22. The method of  claim 21 , wherein the positional domain of each species of capture probe comprises a unique barcode sequence. 
     
     
       23. The method of  claim 1 , wherein the capture probes further comprise a universal domain which is 5′ relative to the capture domainor, if present the positional domain, wherein said universal domain comprises:
 (i) an amplification domain, for amplifying the generated DNA cDNA molecules; and/or 
 (ii) a cleavage domain for releasing the generated DNA cDNA molecules from the surface of the object substrate. 
 
     
     
       24. The method of  claim 1 , wherein the capture domain comprises either (a) a poly-T or poly-U DNA oligonucleotide comprising at least 10 deoxythymidine and/or deoxyuridine residues and/ or (b) a random or degenerate oligonucleotide sequence. 
     
     
       25. The method of  claim 1 , wherein the capture probes are directly immobilized on the object substrate surface by their 5′ end. 
     
     
       26. The method of  claim 1 , wherein the capture probes are indirectly immobilized on the object substrate surface by hybridization to a surface probe probes, wherein the capture domain of the capture probes comprises an upstream sequence that is capable of hybridizing to 5′ end of the surface probes that are immobilized on the object substrate. 
     
     
       27. The method of  claim 26 , wherein the surface probes are immobilized to the object substrate surface by their 3′ ends. 
     
     
       28. The method of  claim 26 , wherein the surface probes comprise a sequence that is complementary to:
 (i) at least part of the capture domain; and 
 (ii) at least part of the a universal amplification domain. 
 
     
     
       29. The method of  claim 28 , wherein the surface probes further comprise a sequence that is complementary to the a positional domain. 
     
     
       30. The method of  claim 1 , wherein the object substrate is composed of a material selected from the group consisting of glass, silicon, poly-L-lysine coated material, nitrocellulose, polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPS), polypropylene, polyethylene and polycarbonate. 
     
     
       31. The method of  claim 1 , wherein the tissue sample is a tissue section or a cell suspension. 
     
     
       32. The method of  claim 1 , wherein the object substrate comprises at least one positional marker to enable orientation of the tissue sample section on the object substrate. 
     
     
       33. The method of  claim 32 , wherein the positional marker is capable of hybridizing to a labelled marker nucleic acid molecule. 
     
     
       34. The method of  claim 32 , wherein the positional marker is capable of hybridizing to a fluorescently labelled marker nucleic acid molecule. 
     
     
       35. The method of  claim 1 , wherein the tissue sample section is imaged using light, bright field, dark field, phase contrast, fluorescence, reflection, interference or confocal microscopy or a combination thereof. 
     
     
       36. The method of  claim 35 , wherein the tissue sample section is imaged using fluorescence microscopy. 
     
     
       37. The method of  claim 1 , comprising a step of modifying the tissue sample prior to the step of contacting the tissue sample with the substrate and/or section prior to the step of generating the cDNA molecules on the object substrate. 
     
     
       38. The method of  claim 37 , wherein the step of modifying the tissue sample comprises dissecting the tissue sample. 
     
     
       39. The method of  claim 38 , wherein the tissue sample is dissected using laser capture microdissection (LCM). 
     
     
       40. The method of  claim 1 , wherein the object substrate is an array substrate that is suitable for use as a sequencing platform. 
     
     
       41. The method of  claim 40 , wherein the object substrate is an array substrate that is suitable for use in next generation sequencing technologies. 
     
     
       42. The method of  claim 1 , wherein the capture probe is immobilized on the object substrate by bridge amplification. 
     
     
       43. The method of  claim 1 , wherein the object substrate is a bead array. 
     
     
       44. A method for spatial detection of a nucleic acid in a tissue section, comprising:
 (a) contacting a tissue section with an object substrate, wherein the object substrate comprises a capture probe comprising a capture domain and a positional domain, wherein the positional domain comprises a plurality of nucleotides that identify a unique location of the capture probe on the object substrate;   (b) allowing a nucleic acid of the tissue section to directly hybridise to the capture probe immobilized on the object substrate under a set of conditions, thereby forming a captured nucleic acid molecule;   (c) generating an amplified nucleic acid molecule from the captured nucleic acid molecule on the object substrate;   (d) labelling the amplified nucleic acid molecule on the object substrate generated in step (c), wherein said labelling step is contemporaneous with said generating step; and   (e) detecting a signal from the labelled amplified nucleic acid molecule on the object substrate,   thereby spatially detecting the nucleic acid in the tissue section.    
     
     
       45. The method of  claim 44 , wherein the nucleic acid of the tissue section comprises RNA.  
     
     
       46. The method of  claim 44 , wherein the nucleic acid of the tissue section comprises mRNA.  
     
     
       47. The method of  claim 44 , further comprising imaging the tissue section.  
     
     
       48. The method of  claim 47 , wherein the tissue section is imaged before step (c).  
     
     
       49. The method of  claim 44 , wherein the detecting comprises sequencing.  
     
     
       50. The method of  claim 49 , wherein the detecting comprises in situ sequencing on the object substrate.  
     
     
       51. The method of  claim 44 , wherein the tissue section is disposed in a gel matrix.  
     
     
       52. The method of  claim 44 , wherein a two-dimensional spatial pattern of members of the captured nucleic acid molecules is preserved.  
     
     
       53. The method of  claim 44 , wherein the object substrate comprises a slide or chip.  
     
     
       54. The method of  claim 44 , wherein the capture probe is oriented to have a free 3′ end to enable the capture probe to function as a reverse transcriptase (RT) primer.  
     
     
       55. The method of  claim 44 , wherein the positional domain is 5′ relative to the capture domain.  
     
     
       56. The method of  claim 1 , further comprising imaging the tissue section before step (c).  
     
     
       57. The method of  claim 1 , further comprising imaging the tissue section after step (c).