Method and product for localized or spatial detection of nucleic acid in a tissue sample

Abstract

Localized detection of RNA in a tissue sample that includes cells is accomplished on an array. The array include a number of features on a substrate. Each feature includes a different capture probe immobilized such that the capture probe has a free 3′ end. Each feature occupies a distinct position on the array and has an area of less than about 1 mm 2 . Each capture probe is a nucleic acid molecule, which includes a positional domain including a nucleotide sequence unique to a particular feature, and a capture domain including a nucleotide sequence complementary to the RNA to be detected. The capture domain can be at a position 3′ of the positional domain.

What is claimed is: 
     
         1 . A method for localized detection of RNA in a tissue sample comprising cells, said method comprising:
 (a) providing an array comprising a plurality of features on a substrate, each feature comprising a different capture probe immobilized thereon such that the capture probe has a free 3′ end, each feature occupying a distinct position on the array and having an area of less than about 1 mm 2 , each capture probe consisting of a nucleic acid molecule comprising the following domains oriented 5′ to 3′:   (i) a positional domain comprising a nucleotide sequence unique to a particular feature; and   (ii) a capture domain comprising a nucleotide sequence complementary to the RNA to be detected;   (b) contacting said array with the tissue sample comprising cells such that the tissue sample contacts a plurality of the features at their distinct positions on the array;   (c) hybridizing the RNA present in the tissue sample comprising cells that are complementary to the capture sequences of the capture probes immobilized on the features, such that the RNA is captured by the capture domain of the capture probes in the features;   (d) generating cDNA molecules from the captured RNA, by extending the capture probes enzymatically using the captured RNA as an extension template, such that the cDNA molecules comprise the nucleotide sequences of the positional domains;   (e) releasing at least part of the cDNA molecules from the features of the surface of the array, and   (f) identifying nucleotide sequences of the positional domain or sequences complementary to the nucleotide sequences of the positional domain present in the released cDNA molecules, wherein the presence of the nucleotide sequence of the positional domain unique to a given particular feature or the sequence complementary to the nucleotide sequence of the positional domain unique to said particular feature indicates that the released cDNA molecule was obtained from RNA present in the tissue sample comprising cells at the distinct position where the tissue sample comprising cells contacted said particular feature.   
     
     
         2 . The method of  claim 1 , further comprising a step of generating a complementary strand of the cDNA molecules. 
     
     
         3 . The method of  claim 2 , wherein the step of releasing at least part of the cDNA molecules from the surface of the array comprises releasing the complementary strands of the cDNA molecules by denaturation. 
     
     
         4 . The method of  claim 3  further comprising a step of amplifying the released complementary strands of the cDNA molecules. 
     
     
         5 . The method of  claim 1  further comprising a step of amplifying the cDNA molecules such that the amplified cDNA molecules comprise nucleotide sequences of the positional domains and nucleotide sequences complementary to the positional sequences. 
     
     
         6 . The method of  claim 5 , wherein said step of amplifying the cDNA molecules functions as the step of releasing at least part of the cDNA molecules from the features of the surface of the array. 
     
     
         7 . The method of  claim 1 , wherein each capture probe consisting of a nucleic acid molecule comprises the following domains oriented 5′ to 3′:
 (i) a cleavage domain; 
 (ii) a positional domain comprising a nucleotide sequence unique to a particular feature; and 
 (iii) a capture domain comprising a nucleotide sequence complementary to the RNA to be detected, 
 and wherein the step of releasing at least part of the cDNA molecules from the features of the surface of the array comprises cleaving the cleavage domain. 
 
     
     
         8 . The method of  claim 7 , wherein the step of cleaving the cleavage domain comprises cleaving the cleavage domain with a cleavage enzyme that recognizes a nucleotide sequence in the cleavage domain and cleaves the cDNA molecules at a position that is 5′ to the positional domain. 
     
     
         9 . The method of  claim 1 , wherein the RNA is selected from the list consisting of mRNA, tRNA, rRNA, viral RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), ribozymal RNA, antisense RNA and non-coding RNA. 
     
     
         10 . The method of  claim 9 , wherein the RNA is mRNA and the capture domain is designed for the selective capture of mRNA. 
     
     
         11 . The method of  claim 10 , wherein the capture domain that is designed for the selective capture of mRNA hybridizes to the poly-A tail of mRNA. 
     
     
         12 . The method of  claim 10 , wherein the domain that is designed for the selective capture of mRNA comprises a poly-T sequence. 
     
     
         13 . The method of  claim 1 , wherein the capture domain comprises a random hexamer sequence. 
     
     
         14 . The method of  claim 1 , wherein step (f) comprises sequencing the released cDNA molecules. 
     
     
         15 . The method of  claim 14  further comprising a step of correlating the sequence analysis information obtained in step (f) with an image of said tissue sample comprising cells, wherein the method includes a step of imaging the tissue sample comprising cells after step (b). 
     
     
         16 . The method of  claim 1 , further comprising determining which genes are expressed at a particular distinct location of the tissue sample comprising cells by a method comprising determining the sequences of the released cDNA molecules comprising the same nucleotide sequence of a positional domain or sequence complementary the nucleotide sequence of a positional domain. 
     
     
         17 . The method of  claim 1 , further comprising determining where a particular gene is expressed in the tissue sample comprising cells by a method comprising identifying the released cDNA molecules comprising a sequence associated with said particular gene and determining which nucleotide sequences of the positional domains or sequences complementary the nucleotide sequences of the positional domains are attached thereto. 
     
     
         18 . The method of  claim 1 , further comprising correlating the nucleotide sequence of a positional domain unique to a given particular feature or the sequence complementary to the nucleotide sequence of a positional domain unique to said particular feature present in the released cDNA molecules to a position in the tissue sample. 
     
     
         19 . The method of  claim 1 , wherein the tissue sample comprising cells is a tissue section or a cell suspension. 
     
     
         20 . The method of  claim 1 , wherein capture probes are immobilized on the substrate by a chemical linker. 
     
     
         21 . The method of  claim 1 , wherein the array is a bead array and the capture probes are immobilized on the beads of the array.