Multivariate Diagnostic Assays and Methods for Using Same
The application describes compositions and methods for detecting the relative expressions of a plurality of target nucleic acid molecules in one assay. The compositions comprise a plurality of probe molecules which specifically bind to one target nucleic acid molecule of a plurality of target nucleic acids in a sample, and a plurality of reference molecules that represent each of the plurality of target nucleic acid molecules, where the probe molecules specifically bind to the plurality of reference molecules, and each of the plurality of reference molecules is present in known amounts in the composition.
1 . A composition for the multiplexed detection of a plurality of target nucleic acid molecules from a biological sample comprising:
a plurality of probe molecules, wherein each probe molecule in the plurality specifically binds to one target nucleic acid molecule in the sample, and wherein the plurality of probe molecules are capable of non-enzymatic direct detection of the target nucleic acid molecules; and,
a plurality of reference molecules that represent each of the plurality of target nucleic acid molecules, wherein the probe molecules specifically bind to the plurality of reference molecules, and wherein each of the plurality of reference molecules is present in known amounts.
2 . The composition of claim 1 , wherein the plurality of reference molecules that represent each of the plurality of nucleic acid molecules comprise synthesized nucleic acids.
3 . The composition of claim 2 , wherein the plurality of synthesized reference molecules that represent each of the plurality of nucleic acid molecules comprise in vitro transcribed RNA.
4 . The composition of claim 2 , wherein the plurality of synthesized reference molecules that represent each of the plurality of nucleic acid molecules comprise chemically synthesized nucleic acids.
5 . The composition of claim 1 , wherein the reference molecules are used to correct for variations in efficiency of an individual assay.
6 . The composition of claim 1 , wherein the plurality of probe molecules comprises about 8 to about 50 probe molecules.
7 . The composition of claim 1 , wherein the plurality of probe molecules comprises about 25 to about 50 probe molecules.
8 . The composition of claim 1 , wherein the plurality of probe molecules comprises about 50 to about 100 probe molecules.
9 . The composition of claim 1 , wherein the plurality of probe molecules comprises more than 100 probe molecules.
10 . The composition of claim 1 , wherein the probe molecules are nucleic acid probes.
11 . The composition of claim 10 , wherein each nucleic acid probe comprises
(i) a target-specific region that specifically binds to a target nucleic acid molecule; and
(ii) a region comprising a plurality of label-attachment regions linked together, wherein each label attachment region is attached to a plurality of label monomers that create a unique code for each target-specific probe, said code having a detectable signal that distinguishes one nucleic acid probe which binds to a first target nucleic acid from another nucleic acid probe that binds to a different second target nucleic acid molecule.
12 . The composition of claim 11 , wherein the plurality of label-attachment regions comprises at least four label attachment regions.
13 . The composition of claim 11 , wherein the plurality of label monomers comprises at least 4 label monomers.
14 . The composition of claim 11 , wherein each of said label monomers are selected from the group consisting of a fluorochrome moiety, a fluorescent moiety, a dye moiety and a chemiluminescent moiety.
15 . The composition of claim 10 , wherein the nucleic acid probe further comprises an affinity tag.
16 . A kit comprising the composition of claim 1 and instructions for the multiplexed detection of a plurality of target nucleic acid molecules.
17 . The kit of claim 16 , further comprising an apparatus, wherein said apparatus comprises a surface capable of binding the hybridized probe molecules of said kit under suitable binding conditions.
18 . The kit of claim 16 , further comprising a composition for the extraction of the target nucleic acids from a biological sample.
19 . The kit of claim 16 , further comprising a reagent selected from the group consisting of a hybridization reagent, a purification reagent, an immobilization reagent and an imaging reagent.
20 . A method of detecting the expression of a plurality of target nucleic acid molecules from a biological sample comprising:
providing a biological sample;
providing a plurality of probe molecules, wherein each probe molecule in the plurality specifically binds to one target nucleic acid molecule in the sample;
contacting the biological sample and the plurality of probe molecules under conditions; sufficient for hybridization of at least one probe molecule and one target nucleic acid molecule; and
detecting a signal associated with each of the plurality of probe molecules bound to each corresponding target nucleic acid molecule, wherein the detection is non-enzymatic.
21 . The method of claim 20 , further comprising
providing a plurality of reference molecules that represent each of the plurality of target nucleic acid molecules, wherein each of the plurality of reference molecules is present in known amounts;
detecting a signal associated with each of the plurality of probe molecules bound to each corresponding reference nucleic acid molecule; and
normalizing the signal associated with each of the plurality of probe molecules bound to each corresponding target nucleic acid molecule with the corresponding signal associated with each of the plurality of probe molecules bound to each corresponding reference nucleic acid molecule,
thereby quantifying the normalized expression of the plurality of target nucleic acid molecules.
22 . The method of claim 21 , wherein the plurality of reference molecules that represent each of the plurality of nucleic acid molecules comprise synthesized nucleic acids.
23 . The method of claim 22 , wherein the plurality of synthesized reference molecules that represent each of the plurality of nucleic acid molecules comprise in vitro transcribed RNA.
24 . The method of claim 22 wherein the plurality of synthesized reference molecules that represent each of the plurality of nucleic acid molecules comprise chemically synthesized nucleic acids.
25 . The method of claim 21 , wherein the reference molecules are used to correct for variations in efficiency of an individual assay.
26 . The method of claim 20 , wherein the plurality of probe molecules comprises about 8 to about 50 probe molecules.
27 . The method of claim 20 , wherein the plurality of probe molecules comprises about 25 to about 50 probe molecules.
28 . The method of claim 20 , wherein the plurality of probe molecules comprises about 50 to about 100 probe molecules.
29 . The method of claim 20 , wherein the plurality of probe molecules comprises more than 100 probe molecules.
30 . The method of claim 20 , wherein the probe molecules are nucleic acid probes.
31 . The method of claim 30 , wherein each nucleic acid probe comprises
(i) a target-specific region that specifically binds to a target nucleic acid molecule; and
(ii) a region comprising a plurality of label-attachment regions linked together, wherein each label attachment region is attached to a plurality of label monomers that create a unique code for each target-specific probe, said code having a detectable signal that distinguishes one nucleic acid probe which binds to a first target nucleic acid from another nucleic acid probe that binds to a different second target nucleic acid molecule.
32 . The method of claim 31 , wherein the plurality of label-attachment regions comprises at least four label attachment regions.
33 . The method of claim 31 , wherein the plurality of label monomers comprises at least 4 label monomers.
34 . The method of claim 31 , wherein each of said label monomers are selected from the group consisting of a fluorochrome moiety, a fluorescent moiety, a dye moiety and a chemiluminescent moiety.
35 . The method of claim 30 , wherein the nucleic acid probe further comprises an affinity tag.
36 . The method of claim 20 , wherein the biological sample is a tissue or cell sample.
37 . The method of claim 20 , wherein the biological sample is a tumor sample.
38 . The method of claim 37 , wherein the tumor sample is a breast tissue sample.
39 . The method of claim 20 , wherein the biological sample is a formalin-fixed paraffin-embedded tissue sample.
40 . The method of claim 20 , wherein the signal is detected without target nucleic acid amplification.