Methods of detecting analytes
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.
1. A method for detecting mRNA from a tissue section, wherein the method comprises:
(a) providing an array, wherein the array comprises a plurality of features on a substrate, wherein each feature occupies a distinct position on the array, and wherein each feature comprises a plurality of capture probes that are directly or indirectly immobilized on the feature, wherein the plurality of capture probes comprise the following domains:
(i) a positional domain comprising a nucleotide sequence unique to the feature; and
(ii) a capture domain comprising a nucleotide sequence for the capture of mRNA;
(b) permeabilizing the tissue section to release mRNA from the tissue section;
(c) hybridizing released mRNA from the tissue section to the capture domains of the capture probes;
(d) extending the capture probes using the hybridized mRNA as an extension template to generate cDNA; wherein the cDNA is tagged by the positional domain;
(e) generating second strand cDNA using a template switching primer;
(f) releasing the second strand cDNA from the features of the array;
(g) determining the sequence of the second strand cDNA, or a complement thereof, and
(h) correlating the sequence of the second strand cDNA, or the complement thereof, to a location in the tissue section.
2. The method of claim 1 , wherein the capture domain comprises a poly-T sequence.
3. The method of claim 2 , wherein the poly-T sequence comprises at least 10 deoxythymidine residues.
4. The method of claim 1 , wherein the positional domain comprises less than 70%, sequence identity across a substantial portion of the mRNAs in the tissue section.
5. The method of claim 1 , wherein the extending step is performed by reverse transcription.
6. The method of claim 1 , wherein the extending step is performed by a reverse transcriptase enzyme.
7. The method of claim 1 , wherein the determining step comprises sequencing.
8. The method of claim 1 , wherein the tissue section is imaged prior to step (b).
9. The method of claim 1 , wherein the array comprises an area of less than 10 cm 2 .
10. The method of claim 1 , wherein the array comprises at least 50,000 features.
11. The method of claim 1 , wherein the array comprises at least 100,000 features.
12. The method of claim 1 , wherein the mRNA comprises polyadenylated RNA.
13. The method of claim 1 , wherein the permeabilization step comprises enzymatic permeabilization.
14. The method of claim 1 , wherein the released second strand cDNA is amplified prior to the determining step.
15. The method of claim 1 , wherein the tissue section is fixed prior to step (b).
16. The method of claim 1 , wherein the tissue section comprises a fixed tissue section.
17. The method of claim 16 , wherein the fixed tissue section is a paraffin-embedded formalin fixed tissue section.
18. The method of claim 16 , wherein the fixed tissue section is a formalin fixed tissue section.
19. The method of claim 1 , wherein the tissue section comprises a fresh frozen tissue section.
20. A method for spatially tagging mRNA from a tissue section, wherein the method comprises:
(a) providing an array, wherein the array comprises a plurality of features on a substrate, wherein each feature occupies a distinct position on the array, and wherein each feature comprises a plurality of capture probes that are directly or indirectly immobilized at the feature, wherein the plurality of capture probes comprise the following domains:
(i) a positional domain comprising a nucleotide sequence unique to the feature; and
(ii) a capture domain comprising a nucleotide sequence for the capture of mRNA;
(b) permeabilizing the tissue section to release mRNA from the tissue section;
(c) hybridizing released mRNA from the tissue section to the capture domains of the plurality of capture probes; and
(d) extending the plurality of capture probes using the hybridized mRNA as an extension template to generate cDNA; wherein the cDNA is tagged by the positional domain;
(e) generating second strand cDNA using a template switching primer; and
(f) releasing the second strand cDNA from the features of the array.
21. The method of claim 20 , wherein the capture domain comprises a poly-T sequence.
22. The method of claim 20 , wherein the extending step is performed by reverse transcription.
23. The method of claim 20 , wherein the array comprises at least 50,000 features.
24. The method of claim 20 , wherein the permeabilization step comprises enzymatic permeabilization.
25. The method of claim 20 , wherein the tissue section is fixed prior to step (b).
26. The method of claim 20 , wherein the tissue section comprises a fixed tissue section.
27. The method of claim 26 , wherein the fixed tissue section is a paraffin-embedded formalin fixed tissue section.
28. The method of claim 26 , wherein the fixed tissue section is a formalin fixed tissue section.
29. The method of claim 20 , wherein the tissue section comprises a fresh frozen tissue section.
30. A method for detecting mRNA in a tissue section, wherein the method comprises:
(a) providing an array, wherein the array comprises a plurality of features on a substrate, wherein each feature occupies a distinct position on the array, and wherein each feature comprises a plurality of capture probes that are directly or indirectly immobilized on the feature, wherein the plurality of capture probes comprise the following domains:
(i) a positional domain comprising a nucleotide sequence unique to the feature; and
(ii) a capture domain comprising a nucleotide sequence for the capture of mRNA;
(b) permeabilizing the tissue section to release mRNA from the tissue section;
(c) hybridizing released mRNA from the tissue section to the capture domains of the plurality of capture probes; (d) extending the capture probes using the hybridized mRNA as an extension template to generate cDNA; wherein the cDNA is tagged by the positional domain;
(e) introducing a polynucleotide tail to the cDNA by terminal transferase activity;
(f) generating a complementary strand of the tagged cDNA;
(g) releasing at least part of the tagged cDNA and/or the complement thereof, from the features of the array; wherein the part includes the positional domain, or a complement thereof;
(h) determining the sequence of the tagged cDNA, or the complement thereof; and
(i) correlating the sequence of the tagged cDNA, or the complement thereof, to a location in the tissue section.
31. The method of claim 30 , wherein the capture domain comprises a poly-T sequence.
32. The method of claim 31 , wherein the poly-T sequence comprises at least 10 deoxythymidine residues.
33. The method of claim 30 , wherein the positional domain comprises less than 70%, sequence identity across a substantial portion of the mRNAs in the tissue section.
34. The method of claim 30 , wherein the extending step is performed by reverse transcription.
35. The method of claim 30 , wherein the extending step is performed by a reverse transcriptase enzyme.
36. The method of claim 30 , wherein the determining step comprises sequencing.
37. The method of claim 30 , wherein the tissue section is imaged prior to step (b).
38. The method of claim 30 , wherein the array comprises an area of less than 10 cm 2 .
39. The method of claim 30 , wherein the array comprises at least 50,000 features.
40. The method of claim 30 , wherein the array comprises at least 100,000 features.
41. The method of claim 30 , wherein the mRNA comprises polyadenylated RNA.
42. The method of claim 30 , wherein the permeabilization step comprises enzymatic permeabilization.
43. The method of claim 30 , wherein the tissue section is fixed prior to step (b).
44. The method of claim 30 , wherein the tissue section comprises a fixed tissue section.
45. The method of claim 44 , wherein the fixed tissue section is a paraffin-embedded formalin fixed tissue section.
46. The method of claim 44 , wherein the fixed tissue section is a formalin fixed tissue section.
47. The method of claim 30 , wherein the tissue section comprises a fresh frozen tissue section.