| CPC C12Q 1/6841 (2013.01) [C12N 15/1065 (2013.01); G01N 21/6428 (2013.01); G01N 21/6458 (2013.01); G02B 21/06 (2013.01); G02B 21/16 (2013.01); G01N 2201/068 (2013.01); G01N 2201/10 (2013.01)] | 30 Claims |
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1. A method, comprising steps of:
(a) performing a first contacting step that comprises contacting a cell comprising a plurality of transcripts with a first plurality of detectably labeled oligonucleotides, each of which targets a transcript and is labeled with a detectable moiety, so that the first plurality comprises at least:
(i) a first oligonucleotide targeting a first transcript in the cell labeled with a first detectable moiety; and
(ii) a second oligonucleotide targeting a second transcript in the cell labeled with a second detectable moiety;
(b) imaging the cell, after the first contacting step so that interaction by hybridizations of oligonucleotides of the first plurality with their target sequences are detected;
(c) performing a second contacting step that comprises contacting the cell, with a second plurality of detectably labeled oligonucleotides, which second plurality includes oligonucleotides targeting transcripts that are targeted by the first plurality so that the second plurality comprises at least:
(i) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, targeting the first transcript; and
(ii) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, targeting the second transcript, wherein the second plurality differs from the first plurality in that at least one of the oligonucleotides present in the second plurality is labeled with a different detectable moiety than the corresponding oligonucleotide targeting the same transcript in the first plurality, so that, in the second plurality:
(iii) the third oligonucleotide is labeled with the first detectable moiety, the second detectable moiety or a third detectable moiety; and
(iv) the fourth oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, the third detectable moiety, or a fourth detectable moiety, wherein either the third oligonucleotide is labeled with a different detectable moiety than was the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than was the second oligonucleotide, or both;
(d) imaging the cell after the second contacting step so that interaction by hybridization of oligonucleotides of the second plurality with their target sequences is detected; and
(e) optionally repeating the contacting and imaging steps, each time with a new plurality of detectably labeled oligonucleotides comprising oligonucleotides that target transcripts targeted by the first and second pluralities, wherein each utilized plurality differs from each other utilized plurality, due to at least one difference in detectable moiety labeling of oligonucleotides targeting the same transcript,
so that the detected signals barcode the first target transcript, and the second target transcript in the cell, wherein the target transcripts can be differentiated by a difference in their barcodes.
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