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 an mRNA from a tissue section, wherein the method comprises:
(a) providing an array comprising a plurality of features on a substrate, wherein each feature occupies a distinct position on the array, wherein each feature comprises a plurality of capture probes, and wherein a capture probe of the plurality of capture probes on each feature comprises a nucleic acid molecule having the following domains:
(i) a positional domain comprising a nucleotide sequence unique to the feature; and
(ii) a capture domain comprising a nucleotide sequence complementary to the mRNA;
(b) hybridizing the mRNA to the capture domain of the capture probe on one or more features of the array;
(c) extending the capture probe using the hybridized mRNA to generate a nucleic acid molecule that comprises a cDNA and the positional domain;
(d) introducing an amplification domain to the nucleic acid molecule using a template switching primer;
(e) generating a second strand that comprises a complement of the cDNA or a portion thereof;
(f) determining a nucleotide sequence of i) at least a portion of the second strand or a complement thereof, and ii) the positional domain, or a complement thereof; and
(g) using the determined nucleotide sequences of at least a portion of the second strand or the complement thereof, and the positional domain, or the complement thereof, to detect the mRNA in the tissue section.
2. The method of claim 1 , wherein the tissue section comprises a fresh-frozen tissue section.
3. The method of claim 1 , wherein the tissue section comprises a formalin-fixed paraffin embedded tissue section.
4. The method of claim 1 , wherein the plurality of features comprises beads.
5. The method of claim 1 , wherein the array comprises at least 50,000 features.
6. The method of claim 4 , wherein the array comprises at least 50,000 beads, wherein each bead comprises an area of about 75 μm 2 .
7. The method of claim 1 , wherein the plurality of capture probes is directly or indirectly immobilized to the features of the array.
8. The method of claim 1 , wherein the method further comprises removing the hybridized mRNA from the cDNA.
9. The method of claim 1 , wherein the nucleic acid molecule is orientated such that the positional domain is 5′ to the capture domain.
10. The method of claim 1 , wherein the capture domain comprises a poly-T sequence.
11. The method of claim 10 , wherein the poly-T sequence comprises at least 25 nucleotides or at least 30 nucleotides.
12. The method of claim 1 , wherein determining the nucleotide sequence of i) at least a portion of the second strand or the complement thereof, and ii) the positional domain, or the complement thereof, comprises sequencing.
13. The method of claim 1 , wherein extending the capture probe comprises a reverse transcription reaction.
14. The method of claim 1 , wherein the capture probe further comprises a cleavage domain.
15. The method of claim 1 , further comprising after step (c), removing unextended capture probes, optionally with an exonuclease.
16. A method for detecting mRNA in a tissue section, wherein the method comprises:
(a) providing an array comprising a plurality of features on a substrate, wherein each feature occupies a distinct position on the array, wherein each feature comprises a plurality of capture probes, and wherein a capture probe of the plurality of capture probes on each feature comprises a nucleic acid molecule having the following domains:
(i) a positional domain comprising a nucleotide sequence unique to the feature; and
(ii) a capture domain comprising a poly-T sequence;
(b) applying the tissue section to the array;
(c) hybridizing one or more mRNAs from the tissue section to the capture domain of one or more capture probes on one or more features of the array;
(d) extending at least one capture probe of the one or more capture probes using a hybridized mRNA of the one or more mRNAs to generate a nucleic acid molecule that comprises a cDNA and the positional domain;
(e) introducing a polynucleotide tail to the 3′ end of the nucleic acid molecule that comprises the cDNA and the positional domain;
(f) removing the tissue section from the array;
(g) generating a second strand that comprises a complement of the cDNA or a portion thereof;
(h) determining a nucleotide sequence of i) at least a portion of the second strand, or the complement thereof, and ii) the positional domain, or a complement thereof; and
(i) using the determined nucleotide sequences of i) at least a portion of the second strand, or the complement thereof, and ii) the positional domain, or the complement thereof, to detect mRNA in the tissue section.
17. The method of claim 16 , wherein the tissue section comprises a fresh-frozen tissue section.
18. The method of claim 16 , wherein the tissue section comprises a formalin-fixed paraffin embedded tissue section.
19. The method of claim 16 , wherein the plurality of features comprises beads.
20. The method of claim 16 , wherein the array comprises at least 50,000 features.
21. The method of claim 19 , wherein the array comprises at least 50,000 beads, wherein each bead comprises an area of about 75 μm 2 .
22. The method of claim 16 , wherein the plurality of capture probes is directly or indirectly immobilized to the features of the array.
23. The method of claim 16 , wherein the poly-T sequence comprises at least 25 nucleotides or at least 30 nucleotides.
24. The method of claim 16 , wherein the nucleic acid molecule is orientated such that the positional domain is 5′ to the capture domain.
25. The method of claim 16 , wherein the capture probe further comprises a cleavage domain.
26. The method of claim 16 , wherein determining the nucleotide sequence of at least a portion of the second strand or the complement thereof, and the positional domain, or the complement thereof, comprises sequencing.
27. The method of claim 16 , wherein removing the tissue section comprises treatment with a proteinase, optionally wherein the proteinase is Proteinase K.
28. The method of claim 16 , wherein extending the at least one capture probe comprises a reverse transcription reaction, and wherein introducing the polynucleotide tail is performed using a reverse transcriptase.
29. The method of claim 16 , wherein the method further comprises hybridizing a template switch primer to the polynucleotide tail, and extending the nucleic acid molecule in step (e) using the template switch primer as a template, wherein the template switch primer comprises an amplification domain, and wherein a complement of the amplification domain is incorporated into the nucleic acid molecule.
30. A method for spatially tagging an mRNA from a tissue section, wherein the method comprises:
(a) providing an array comprising a plurality of features on a substrate, wherein each feature occupies a distinct position on the array, wherein each feature comprises a plurality of capture probes, and wherein a capture probe of the plurality of capture probes on each feature comprises a nucleic acid molecule having the following domains:
(i) a positional domain comprising a nucleotide sequence unique to the feature; and
(ii) a capture domain comprising a nucleotide sequence complementary to the mRNA;
(b) hybridizing the mRNA to the capture domain of one or more capture probes on one or more features of the array;
(c) extending at least one capture probe of the one or more capture probes using the hybridized mRNA to generate a nucleic acid molecule that comprises a cDNA and the positional domain;
(d) introducing an amplification domain to the nucleic acid molecule using a template switching primer; and
(e) generating a second strand that comprises a complement of the cDNA or a portion thereof.
31. The method of claim 30 , wherein the tissue section comprises a fresh-frozen tissue section.
32. The method of claim 30 , wherein the tissue section comprises a formalin-fixed paraffin embedded tissue section.
33. The method of claim 30 , wherein the plurality of features comprises beads, and the array comprises at least 50,000 beads, wherein each bead comprises an area of about 75 μm 2 .
34. The method of claim 30 , wherein the plurality of capture probes is indirectly immobilized to the features of the array.
35. The method of claim 30 , wherein the plurality of capture probes is directly immobilized to the features of the array.
36. The method of claim 30 , wherein the capture domain comprises a poly-T sequence.
37. The method of claim 36 , wherein the poly-T sequence comprises at least 25 nucleotides or at least 30 nucleotides.
38. The method of claim 30 , wherein the method further comprises removing the hybridized mRNA from the capture domain of the capture probe.
39. The method of claim 30 , wherein the capture probe further comprises a cleavage domain.
40. The method of claim 30 , further comprising after step (c), removing unextended capture probes, optionally with an exonuclease.
41. The method of claim 30 , wherein the method further comprises hybridizing a template switch primer to the 3′ end of the nucleic acid molecule.
42. The method of claim 30 , wherein the method further comprises extending the nucleic acid molecule using the template switch primer as a template.