CPC C12Q 1/6837 (2013.01) [C12N 15/1065 (2013.01); C12Q 1/682 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6816 (2013.01); C12Q 1/6827 (2013.01); C12Q 1/6841 (2013.01); C12Q 1/6844 (2013.01); C12Q 1/6876 (2013.01); G01N 1/30 (2013.01); G01N 1/42 (2013.01); G16B 30/00 (2019.02); G16B 50/20 (2019.02); G16B 50/30 (2019.02); C12Y 600/00 (2013.01)] | 47 Claims |
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.
|