Method for detecting chromosome structure and gene expression simultaneously in single cells

An assay for simultaneously determining the structural conformation of a chromosome in a cell and quantitatively measuring the level of gene expression at a chromosomal location, the assay comprising hybridizing a plurality of target sequences of more than one transcribed RNA in the cell with members of a plurality of fluorescently labeled oligonucleotide probe sets and counting fluorescent spots corresponding to single molecules of target transcribed RNAs to obtain a quantitative gene expression profile corresponding to the simultaneously determined, associated chromosomal conformation, wherein:

• (i) each probe set utilizes a distinguishable color;
• (ii) the plurality of targeted sequences of transcribed RNA are transcribed from portions of at least one chromosome;
• (iii) the pattern of fluorescently labeled probes hybridized to the target sequences of transcribed RNA is indicative of the structural conformation of the chromosome;
• (iv) each fluorescently labeled oligonucleotide probe set includes more than sixteen fluorescently labeled oligonucleotides;
• (v) the locations of the hybridized probes are simultaneously indicative of the chromosomal locations of gene expression for the transcribed RNAs, and each of the fluorescently labeled oligonucleotides are coupled to an organic dye at their 3′ end;
• (vi) the number, the intensity, or both, of fluorescing probes is indicative of the level of gene expression at the chromosomal locations;
• (vii) each fluorescently labeled probe targets an intron of the transcribed RNA sequence;
• thereby simultaneously determining the structural conformation of a chromosome in a cell and measuring the level of gene expression at a chromosomal location;
• (viii) at least two introns are targeted in the cell and at least sixteen fluorescently labeled probes having different nucleotide sequences are complementary to each targeted intron;
• and
• (ix) a fluorescent spot is discarded as background based at least on color.