TECHNICAL FIELD

This disclosure generally relates to materials and methods for serial multiplexed detection of nucleic acids in cells and tissues.

BACKGROUND

Ribonucleic acid (RNA) is the product of gene transcription from deoxynucleic acid (DNA) that forms the template for protein biosynthesis. In addition to their role in protein translation, it is well known that nucleic acids molecules exhibit other diverse biological functions. Thus, the expression level and molecular identity of nucleic acid species needs to be measured with a high level of spatial resolution in their tissue of origin in order to understanding the relationship between gene expression and cellular phenotype. This is particularly true in complex heterogeneous tissues such as, for example, the nervous system, where numerous cell types can be distinguished by different gene expression profiles and whose functional characteristics appear to be an essential component of the complexity of the brain.

SUMMARY

Materials and methods for effectively performing serial multiplexed FISH analysis are provided herein.

In one aspect, a method of analyzing the RNA in a cell or a tissue is provided. Such a method typically includes contacting a cell or a tissue with a first DNA probe set, wherein the first DNA probe set binds to a first set of RNAs; contacting the first probe set-bound cell or tissue with RNase H to remove the first probe set; contacting the cell or the tissue with a second probe set, wherein the second DNA probe set binds to a second set of RNAs. In some embodiments, the contacting step is by a branched DNA (bDNA) RNA-single molecule (sm) fluorescent in situ hybridization (FISH) method.

In some embodiments, such a method further includes contacting the second probe set-bound cell or tissue with RNase H to remove the second probe set; and contacting the cell or the tissue with a third probe set, wherein the third DNA probe set binds to a third set of RNAs.

In some embodiments, such a method further includes contacting the third probe set-bound cell or tissue with RNase H to remove the third probe set; and contacting the cell or the tissue with a fourth probe set, wherein the fourth DNA probe set binds to a fourth set of RNAs.

In some embodiments, such a method further includes contacting the fourth probe set-bound cell or tissue with RNase H to remove the fourth probe set; and contacting the cell or the tissue with a fifth probe set, wherein the fifth DNA probe set binds to a fifth set of RNAs.

In some embodiments, such a method further includes washing the cell or the tissue after the RNase H contacting step to remove the removed probe set and the RNase H. In some embodiments, such a method further includes imaging the cell or the tissue after the probe set contacting step.

In some embodiments, the DNA probe set includes at least one DNA oligonucleotide probe. In some embodiments, the DNA probe set includes a plurality of DNA oligonucleotide probes. In some embodiments, each DNA oligonucleotide probe in the DNA probe set includes a differentially detectable label.

In one aspect, a method of analyzing the RNA in a cell or a tissue is provided. Such a method typically includes contacting a cell or a tissue with a first DNA probe set, wherein the first DNA probe set binds to a first set of RNAs; contacting the first probe set-bound cell or tissue with DNase to remove the first probe set; contacting the cell or the tissue with a second probe set, wherein the second DNA probe set binds to a second set of RNAs. In some embodiments, the contacting step is by a branched DNA (bDNA) RNA-single molecule (sm) fluorescent in situ hybridization (FISH) method. In some embodiments, such a method can further include washing the cell or the tissue after the DNase contacting step to remove the removed probe set and the DNase.

In some embodiments, such a method can further include contacting the second probe set-bound cell or tissue with DNase to remove the second probe set; and contacting the cell or the tissue with a third probe set, wherein the third DNA probe set binds to a third set of RNAs. In some embodiments, such a method can further include contacting the third probe set-bound cell or tissue with DNase to remove the third probe set; and contacting the cell or the tissue with a fourth probe set, wherein the fourth DNA probe set binds to a fourth set of RNAs. In some embodiments, such a method can further include contacting the fourth probe set-bound cell or tissue with DNase to remove the fourth probe set; and contacting the cell or the tissue with a fifth probe set, wherein the fifth DNA probe set binds to a fifth set of RNAs.

In some embodiments, such a method can further include imaging the first probe set-bound cell or tissue. In some embodiments, such a method can further include imaging the second probe set-bound cell or tissue. In some embodiments, such a method can further include aligning the second probe set-bound cell or tissue with the first probe set-bound cell or tissue.

In some embodiments, the DNA probe set includes at least one DNA oligonucleotide probe. In some embodiments, the DNA probe set includes a plurality of DNA oligonucleotide probes. In some embodiments, each DNA oligonucleotide probe in the DNA probe set includes a differentially detectable label.

In another aspect, an article of manufacture is provided. Such an article of manufacture typically includes at least one DNA probe set, wherein the at least one DNA probe set binds to a set of RNAs; and RNase H. In some embodiments, such an article of manufacture further includes reagents for hybridizing, selectively, the at least one DNA probe set to the set of RNAs.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

