U.S. patent application number 17/833279 was filed with the patent office on 2022-09-22 for materials and methods for serial multiplexed detection of rna in cells and tissues.
The applicant listed for this patent is Howard Hughes Medical Institute. Invention is credited to Fredrick Henry, Scott Sternson, Shengjin Xu, Hui Yang.
Application Number | 20220298563 17/833279 |
Document ID | / |
Family ID | 1000006388233 |
Filed Date | 2022-09-22 |
United States Patent
Application |
20220298563 |
Kind Code |
A1 |
Sternson; Scott ; et
al. |
September 22, 2022 |
MATERIALS AND METHODS FOR SERIAL MULTIPLEXED DETECTION OF RNA IN
CELLS AND TISSUES
Abstract
This disclosure describes materials and methods for effectively
performing serial multiplexed FISH analysis.
Inventors: |
Sternson; Scott; (Chevy
Chase, MD) ; Henry; Fredrick; (Chevy Chase, MD)
; Yang; Hui; (Chevy Chase, MD) ; Xu; Shengjin;
(Chevy Chase, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Howard Hughes Medical Institute |
Chevy Chase |
MD |
US |
|
|
Family ID: |
1000006388233 |
Appl. No.: |
17/833279 |
Filed: |
June 6, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15724693 |
Oct 4, 2017 |
11352660 |
|
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17833279 |
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62403904 |
Oct 4, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6876 20130101;
C12Q 1/682 20130101; C12Q 1/6841 20130101 |
International
Class: |
C12Q 1/6841 20060101
C12Q001/6841; C12Q 1/6876 20060101 C12Q001/6876; C12Q 1/682
20060101 C12Q001/682 |
Claims
1. An article of manufacture, comprising at least one DNA probe
set, wherein the at least one DNA probe set binds to a set of RNAs;
and RNase H or DNase.
2. The article of manufacture of claim 1, further comprising:
reagents for hybridizing, selectively, the at least one DNA probe
set to the set of RNAs.
3. The article of manufacture of claim 1, further comprising a
second DNA probe set, wherein the second DNA probe set binds to a
second set of RNAs.
4. The article of manufacture of claim 3, further comprising a
third probe set, wherein the third DNA probe set binds to a third
set of RNAs.
5. The article of manufacture of claim 4, further comprising a
fourth probe set, wherein the fourth DNA probe set binds to a
fourth set of RNAs.
6. The article of manufacture of claim 5, further comprising a
fifth probe set, wherein the fifth DNA probe set binds to a fifth
set of RNAs.
7. The article of manufacture of claim 2, wherein the reagents for
hybridizing, selectively, the at least one DNA probe set to the set
of RNAs comprise reagents suitable for use in a branched DNA (bDNA)
RNA-single molecule (sm) fluorescent in situ hybridization (FISH)
method.
8. The article of manufacture of claim 1, wherein the at least one
DNA probe set comprises at least one DNA oligonucleotide probe.
9. The article of manufacture of claim 1, wherein the at least one
DNA probe set comprises a plurality of DNA oligonucleotide
probes.
10. The article of manufacture of claim 9, wherein each of the
plurality of DNA oligonucleotide probes in the at least one DNA
probe set comprises a differentially detectable label.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 15/724,693, filed Oct. 4, 2017, now allowed, which is claims
the benefit of priority under 35 U.S.C. .sctn. 119(e) to U.S.
Application No. 62/403,904 filed Oct. 4, 2016, both of which are
incorporated by reference.
TECHNICAL FIELD
[0002] This disclosure generally relates to materials and methods
for serial multiplexed detection of nucleic acids in cells and
tissues.
BACKGROUND
[0003] 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
[0004] Materials and methods for effectively performing serial
multiplexed FISH analysis are provided herein.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
DESCRIPTION OF DRAWINGS
[0017] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0018] FIG. 1 is a schematic showing the branched DNA (bDNA) smFISH
methods. Adapted from Battich et al., 2013, Nature Methods,
10:1127-33.
[0019] FIG. 2 are photographs that demonstrate ineffective
stripping via warm water exposure. Left panel, bDNA FISH
amplification and binding with AgRP (white) as well as the nuclear
stain, DAPI (blue, 4'6-diamidino-2-phenylindole); Center panel,
bDNA FISH following warm water stripping removes the fluorescent
signal; Right panel, second bDNA FISH amplification and binding.
Schematics above each image correspond to the relevant portions of
the schematic shown in FIG. 1.
[0020] FIG. 3 are photographs that demonstrate ineffective
stripping via photobleaching. Left panel, bDNA FISH amplification
and binding with AgRP; Center panel, bDNA FISH following stripping
by photobleaching; Right panel, second bDNA FISH amplification and
binding. Schematics above each image correspond to the relevant
portions of the schematic shown in FIG. 1.
[0021] FIG. 4 are photographs showing that integrity of the DAPI
nuclear stain is severely compromised after stripping by DNAse.
Because nuclear stain is often used to align tissue sections across
multiple rounds of FISH, this creates computational challenges
after stripping with DNase. Left panel, bDNA FISH amplification and
binding with CRH; Right panel, bDNA FISH following stripping with
DNase.
[0022] FIG. 5 are photographs showing effective removal of bDNA
FISH probes using RNaseH. Left panel, bDNA FISH amplification and
binding with AgRP; Center panel, bDNA FISH following enzymatic
stripping with RNase H; Right panel, second bDNA FISH amplification
and binding. Schematics above each image correspond to the relevant
portions of the schematic shown in FIG. 1; red X's indicate that
the mRNA that was previously bound to the bDNA probe is selectively
digested and, thus, the entire bDNA tree is removed.
[0023] It was demonstrated that, after performing branched DNA/RNA
smFISH, tissue treatment with RNaseH digestion of RNA-DNA hybrids
followed by mild thermal stripping of the remaining branched DNA
hybridization tree led to robust removal of probe-related signal
that did not return after subsequent re-amplification steps. FIG. 5
shows that the smFISH signal for AgRP (left) was effectively
removed after a 20 min exposure to RNaseH at 40.degree. C. to
degrade DNA/RNA complexes (middle; and step 1 in cartoon schematic
above photographs). An additional step involving a 30 min
incubation at 65.degree. C. in H2O (step 2 in cartoon) served to
inactivate RNase H and remove the branched DNA chain (after
degradation of the mRNA/DNA complex), and re-amplification with
AMP1-3 revealed low to undetectable levels of bDNA-related signal,
indicating successful stripping (right).
[0024] FIG. 6 are photographs showing that RNaseH stripping
eliminates mRNA complexed with bDNA probes. Left panel, bDNA FISH
amplification and binding with Gad2; Right panel, bDNA FISH
amplification and binding with Gad2 following stripping with RNase
H.
[0025] FIG. 7 is a photograph showing serial multiplexing using
bDNA smartFISH. Blue, Trh; Green, Pomc; Purple, Tac2; White,
Vglut2; Red, AgRP; Yellow, Vgat.
[0026] FIG. 8 is a photograph showing tissue integrity with IHC
after bDNA smartFISH.
[0027] FIG. 9 are photographs showing that DNase I completely
removes bDNA probes and amplification oligonucleotides. Left panel,
three genes, Avp (green), Sst (red), and Trh (yellow) were detected
with 3-plex RNAscope.RTM., a bDNA-based RNA-FISH platform. Cellular
nuclei are stained with DAPI (blue). Right panel, after DNase I
stripping and subsequent application of amplification
oligonucleotides in the absence of gene specific probes, no FISH
signals were observed. Also, nuclei are no longer reliably stained
with DAPI (imaging settings identical to Right panel).
[0028] FIG. 10 are photographs showing that the same gene can be
stripped and re-probed. The gene Cckar was sequentially probed and
DNase1 stripped in 3 rounds of FISH.
[0029] FIG. 11 is a schematic showing that alignment of a tissue
sample across multiple rounds of FISH requires a reference (R)
tissue marker that can be used to align with cellular resolution. A
fluorescent staining pattern in the tissue that has a consistent
appearance can be simultaneously imaged and used as the reference
with each round of FISH.
[0030] FIG. 12 are images showing the alignment of tissue sections
from each round of FISH using DNase I stripping of bDNA probes.
Left panels, DAPI staining pattern after DNase I stripping, which
is termed DAPI residual. Although nuclear patterns of DAPI staining
are not constant across subsequent rounds of FISH, the high
frequency fibrous signals remain consistent across at least 4
rounds of FISH. Right panels, with DAPI residual in Round 1 FISH as
a reference, each subsequent round of FISH image can be precisely
aligned with the residual DAPI signal from Round 1. The high
frequency fibrous non-nuclear signal in the images is responsible
for successful alignment.
[0031] FIG. 13 are photographs showing four rounds of FISH, with 3
distinct transcripts detected in each round, overlaid on their
corresponding DAPI staining patterns. Left to right: Vglut2
(green), Gad2 (red), and Syp (yellow) were probed in Round 1; Crh
(green), Reln (red), Ntng1 (yellow) were probed in Round 2; Pdyn
(green), Penk (red), Trh (yellow) were probed in Round 3; and Oxy
(green), Avp (red), and Sst (yellow) were probed in Round 4.
DETAILED DESCRIPTION
[0032] A number of techniques have been developed in recent years
to obtain measurements of gene expression on a single cell level.
Sequencing or PCR-based methods, while highly quantitative,
typically require dissociation of tissue sections into homogenous
single cell suspensions for further processing, thus removing
valuable information regarding the spatial arrangements of cells
with unique combinations of gene expression. In contrast to such
methods, image-based readouts of gene expression, including single
molecule fluorescent in situ hybridization (smFISH) of RNA
(sometimes referred to as RNA-smFISH), allows visualization of
individual transcripts in large groups of single cells without
gross disturbance of their spatial arrangement.
[0033] A widely used approach for smFISH of RNA uses a combination
of oligonucleotides labeled with multiple fluorophores to "tile"
the length of an mRNA species of interest. This method, sometimes
referred to as o-nuc smFISH, contains no true signal amplification;
only the linear combination of oligonucleotide-associated
fluorophores that line the transcript of interest.
[0034] A second approach uses multiple pairs of primary DNA probes
designed to hybridize to two adjacent regions, each 20-30
nucleotides in length, at several sites along the length of a
transcript. These combinations of primary probes then provide
hybridization sites for a second pre-amplification probe, which
allows hybridization of multiple amplification probes that serve as
binding regions for fluorescently conjugated DNA molecules (FIG. 1;
adapted from Battich et al., 2013, Nature Methods, 10:1127-33).
FIG. 1 shows signal amplification via the step-wise addition of 1)
sequence-specific primary probes; 2) pre-amplification DNA
molecules; 3) amplification DNA molecules; and 4)
fluorophore-conjugated DNA probes. The resulting tree-like
structure takes on the appearance of a DNA pillar with many
branches, hence the common referral of this method as branched DNA
(bDNA) RNA-smFISH. bDNA RNA-smFISH or variants thereof (e.g.,
MER-FISH) are suitable for use in the methods described herein. For
example, Player et al., 2001, J. Histochem. Cytochem.,
49(5):603-12; Wang et al., 2012, J. Mol. Diag., 14(1):22-9; Battich
et al., 2013, Nature Methods, 10:1127-33; Ke et al., 2013, Nat.
Methods, 10(9):857-60; Lee et al., 2014, Science, 343(6177):1360-3;
Chen et al., 2015, Science, 348(6233):aaa6090; and US 2013/0171621,
all of which are incorporated herein by reference in their
entirety, describe various multiplex assays.
[0035] The additional amplification sites in bDNA-based methods
provide a high signal-to-noise compared with other approaches. A
recent side-by-side comparison indicated that bDNA-labeled mRNA
spots were roughly 100 times ("100.times.") brighter than those
tiled with fluorescently conjugated oligonucleotides (Battich, et
al., 2013, Nature Methods, 10:1127-33). Current commercial
application of bDNA-based methods in conjunction with smFISH allow
simultaneous labeling of up to four mRNA species in a single piece
of tissue via the combined use of separate oligonucleotide
sequences for both the mRNA detection and the associated signal
amplification systems. Current limits on the number of mRNA species
able to be differentiated in this manner are constrained by the
number of oligonucleotide sequences for the branched DNA detection
process as well as spectral separation between the fluorophores
used in the final amplification step.
[0036] The high signal-to-noise readout of bDNA-labeled mRNA
provides a distinct advantage for image-based methods to assess the
presence of genes having a low level of expression, as is often the
case with many important mRNA products in neurons (e.g., encoding
transcription factors, neuropeptide receptors, etc.). However,
there is often a need to visualize the expression of multiple genes
simultaneously ("multiplexing") in a piece of tissue.
[0037] Efforts to perform multiplexed smFISH are typically limited
by the number of independent signals representing distinct
hybridization targets that can be read out during a single imaging
round. To increase multiplexing, and thus, the number of genes
whose transcripts can be quantified in a given cell, techniques
have been developed to permit multiple rounds of mRNA/DNA duplex
hybridization. These techniques include, for example, thermal
stripping of probes bound to in situ mRNA molecules, photobleaching
of fluorophores present on the hybridized molecules, and treatment
with the DNA digesting enzyme, DNase. It has been determined,
however, that these existing methods for serial multiplexed RNA in
situ hybridization are, in practice, not suitable for multiple
rounds of probe hybridization and bDNA signal amplification in
sensitive tissues such as brain.
[0038] Given such difficulties, recent efforts have led to the
development of new methods for removing bDNA probes from previously
labeled tissue, thus permitting multiple rounds of three-plex mRNA
labeling in brain tissue with little to no disturbance of tissue
integrity. This disclosure describes such a method and was used to
achieve multiplexed labeling of more than 9 genes (e.g., more than
10 genes, more than 12 genes, more than 15 genes) in situ with high
signal-to-noise using sequential rounds of bDNA-based RNA-smFISH
and enzymatic stripping of the hybridized RNA transcript. This
method, referred to herein as "smart" (serial multiplexing by
ablation of RNA targets) FISH, allows for reliable ex vivo
detection of genes expressed at low levels, preserves tissue
integrity across imaging rounds, and is suitable for combined use
with other labeling techniques, including immunohistochemical
protein labeling.
[0039] smartFISH provides a new implementation for RNaseH, an
enzyme that selectively hydrolyzes RNA that is hybridized to DNA
and is widely used, for example, in cloning techniques to eliminate
RNA-DNA hybrids. While RNase H has been used previously to degrade
mRNA post-cDNA conversion for the purpose of in situ sequencing,
RNase H has not been used to selectively and repetitively strip
DNA-RNA hybrids during identification of distinct mRNA species in
cells, tissues, organs, or organisms.
[0040] Another approach, referred to as multiFISH, provide an
implementation for DNase (e.g., DNase I) in repeated rounds of
stripping after bDNA RNA-smFISH against mRNA. This method avoids
the modestly elevated temperatures (65.degree. C.) used in the
RNase H stripping method. DNase is an enzyme that catalyzes the
hydrolytic cleavage of phosphodiester bonds in the backbone of the
DNA. While DNase has been used previously for multiplexed FISH, it
has not been applied to bDNA FISH. In addition, prior
implementations of multiplexed FISH using DNase stripping have not
use fully automated alignment of tissue sections after each round
of FISH due to loss binding of a nuclear stain (e.g., DAPI) due to
digestion of nuclear DNA by DNase during the probe stripping
process. In the multiFISH method, tissue labeled with bDNA probes
for RNA-FISH can be stripped with DNase (e.g., 20 Kunitz units of
DNase I in 100 .mu.l of a DNase I buffer, such as RDD buffer from
Quiagen, for 4 h), which can be inactivated, for example, by
washing in phosphate buffered saline (PBS). This method ensures
thorough removal of the probes and permits subsequent
re-probing.
[0041] Multiple rounds of FISH on the same tissue requires
alignment of a reference tissue features across each round. Cells
or tissue treated with RNase H retain bright nuclear staining,
which allows for simple alignment of tissue sections across
multiple rounds of FISH based on aligning the cellular nuclei with
standard image registration programs, such as those available in
the open source image analysis software FIJI. After DNase I
treatment, however, DAPI stains a combination of nuclear and high
frequency fibrous non-nuclear features that are stable across
multiple rounds of FISH. Using these stable DAPI-binding features,
non-linear registration algorithms can be used to align
three-dimensional images of these tissue sections with high
precision by combining three-dimensional affine transformation and
non-linear deformation transformation using, for example the
Advanced Normalization Tools software package. This algorithm
achieves automated computational alignment of cells or tissues
across multiple rounds of FISH images. For example, alignment can
be performed with cellular resolution of a single tissue sample
over at least four rounds of three-plex FISH (i.e., a total of
twelve FISH probes).
[0042] The high signal to noise detection of mRNA molecules present
at a wide range of expression profiles in the smartFISH and
multiFISH protocols described herein, coupled with the degree to
which tissue integrity is preserved, will permit seamless
integration into pipelines involving post-hoc analysis of tissue
that has been previously involved in any number of procedures
including, without limitation, in vivo Ca2+ imaging (Betley et al.,
2015, Nature, 521:180-5; Sofroniew et al., 2016, Elife,
5pii:e14472); fine scale assessment of synaptic connectivity using
large volume array-tomography (Bloss et al., 2016, Neuron,
89(5):1016-30); and cell type identification via molecular
profiling after high density reconstruction with improved methods
for serial two-photon tomography (Economo et al., 2016, Elife,
5:e10566).
[0043] Widespread implementation of the approach described herein
enables the unbiased classification of functionally relevant gene
expression profiles without making complicated and unnecessary
perturbations to the cell or tissue. The procedure is relatively
straightforward, able to be accomplished using commercially
available reagents, and cost-limited only by the number of genes
one wishes to detect in a given experiment. smartFISH and multiFISH
can be incorporated into a wide variety of other imaging approaches
in order to couple gene expression profiles with functional and/or
anatomical data sets. The possibility of assembling these types of
extraordinarily rich multi-modal data sets, as well as the ability
to perform such experiments in any species for which accurate
information has been acquired about the molecular profiles of cells
in a given region, make smartFISH and multiFISH extremely powerful
methods.
[0044] While operating at a lower throughput than methods able to
profile hundreds of mRNAs species in situ, the methodology
described herein will be useful for those looking to assess many
(e.g., 10, 20, 30 or more) transcript types in situ under a very
high signal to noise regime. To the knowledge of the inventors,
there have been no published reports of successful smFISH
multiplexing via sequential hybridization and stripping of bDNA
probes. In addition, the methods described herein have been
successfully demonstrated in complex tissues while other approaches
have been limited to cell culture systems.
[0045] In accordance with the present invention, there may be
employed conventional molecular biology, microbiology, biochemical,
and recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. The invention
will be further described in the following examples, which do not
limit the scope of the methods and compositions of matter described
in the claims.
EXAMPLES
Example 1--Ineffective Stripping Via Warm Water Exposure
[0046] It was determined that, probably because of the stability of
core elements of the branched DNA tree, thermal stripping, using
conditions that were compatible with tissue integrity, was
incomplete. FIG. 2 shows that the smFISH signal for AgRP (left) was
effectively removed after 2 hrs of incubation in H2O at 37.degree.
C. (middle). However, exposing the tissue to rounds 1-3 of
amplification resulted in robust detection of earlier "stripped"
signal (right), indicating the successful removal of the
fluorophore-containing component of the branched DNA chain (AMP3
from FIG. 1) but insufficient removal of the DNA/mRNA complex
itself.
Example 2--Ineffective Stripping Via Light Exposure or
Photobleaching
[0047] It was demonstrated that photobleaching did not prevent
hybridization in later rounds from detecting RNA species from
earlier rounds, presumably because the oligonucleotide with the
conjugated bleached fluorophore unbinds and another
fluorophore-labeled oligonucleotide binds to the remaining branched
DNA tree in the subsequent hybridization round (cartoon schematic
above photographs). FIG. 3 shows that the smFISH signal for AgRP
(left) was effectively removed after a 5 min exposure to high
intensity 405 nm wavelength light (middle). However, exposing the
tissue to rounds 1-3 of amplification resulted in robust detection
of earlier "stripped" signal (right), indicating incomplete removal
of mRNA/DNA probe complexes. Similar results were obtained after
photobleaching using light at 488, 555, or 647 nm (data not
shown).
Example 3--Tissue Integrity Appeared to be Compromised after
Stripping by DNAse
[0048] It also was determined that, while DNase treatment
effectively eliminated smFISH signal after re-amplification, it
also led to a loss of tissue integrity that makes multiple
hybridization events difficult. FIG. 4 shows that the smFISH signal
for CRH (left, white) was effectively removed after hydrolytic
cleavage of phosphodiester bonds in bDNA trees (middle). However,
this enzymatic degradation process caused some amount of damage to
the surrounding tissue, as evidenced by distortion of DAPI staining
of cell nuclei. Because the retention of fine-grained cellularity
in situ is paramount to the success of implementing a detection
strategy involving multiple rounds of labeling, a different
alignment protocol was required when using DNAse (see Example 9
below).
Example 4--Effective Enzymatic Removal of bDNA FISH Probes Using
RNase H
[0049] It was demonstrated that, after performing branched DNA/RNA
smFISH, tissue treatment with RNaseH digestion of RNA-DNA hybrids
followed by mild thermal stripping of the remaining branched DNA
hybridization tree led to robust removal of probe-related signal
that did not return after subsequent re-amplification steps. FIG. 5
shows that the smFISH signal for AgRP (left) was effectively
removed after a 20 min exposure to RNaseH at 40.degree. C. to
degrade DNA/RNA complexes (middle; and step 1 in cartoon schematic
above photographs). An additional step involving a 30 min
incubation at 65.degree. C. in H2O (step 2 in cartoon) served to
inactivate RNase H and remove the branched DNA chain (after
degradation of the mRNA/DNA complex), and re-amplification revealed
low to undetectable levels of mRNA-related signal, indicating
successful stripping (right). This method is referred to herein as
"smartFISH".
Example 5--RNase H Stripping Eliminates mRNA Complexed With bDNA
Probes
[0050] Importantly, attempts to re-probe tissue in order to label
an mRNA species identical to that probed in a previous round
produced no signal after enzymatic stripping, which indicates
near-total elimination of all mRNA species associated with the gene
of interest. FIG. 6 shows that Gad2 mRNA signal, detected via bDNA
RNA-smFISH (left, white), was effectively removed after exposure to
RNaseH. Re-probing for the same gene revealed extremely limited
signal detected using identical imaging conditions (right, white).
These results demonstrated that RNaseH-mediated degradation of
smFISH signal occurred via selective and comprehensive degradation
of mRNA species that were complexed with DNA probes.
Example 6--Serial Multiplexing Using bDNA smartFISH
[0051] After RNaseH stripping, it was shown that multiple
subsequent rounds of three-plex bDNA RNA-smFISH could be performed
with high signal-to-noise ratio and excellent tissue integrity. The
simultaneous labeling of six genes in the mouse hypothalamus was
demonstrated. For ease of viewing, FIG. 7 shows the product of two
rounds of three-plex labeling, although three or more rounds were
performed with virtually undetectable degradation of tissue
integrity.
Example 7--Immunohistochemistry after bDNA smartFISH
[0052] It also was shown that smartFISH was compatible with
immunohistochemical detection with antibodies, providing a means to
combine molecular profiling with protein quantification on a single
cell basis in situ. FIG. 8 shows a region of the mouse hypothalamus
in which mRNA associated with the genes vgat (yellow) and vglut2
(green) were detected using bDNA smartFISH. After enzymatic
stripping using RNaseH, the tissue was then subject to
immunohistochemical detection of the proteins NeuN (blue) and
tyrosine hydroxylase (magenta) using standard methods.
Example 8--Effective Removal of bDNA FISH Probes Using DNase
[0053] A method to achieve multiplexed labeling of .gtoreq.12 genes
in situ is described herein (referred to herein as "multiFISH").
This method uses sequential rounds of RNA-FISH with bDNA-probes and
uses DNase to strip the probes and amplification oligonucleotides.
Unlike the RNase H stripping method described herein, the same gene
can be probed in subsequent FISH rounds. In addition, this method
avoids the elevated temperatures used in the RNase H stripping
method.
[0054] An experimental procedure has been developed for multiple
rounds of bDNA RNA-FISH on the same tissue sample by stripping bDNA
probes and amplification oligonucleotides with DNase I. Recombinant
RNase-free DNase I can be inactivated by washing in phosphate
buffered saline (PBS). Thus, DNase I can be eliminated by PBS
washing after stripping and before the next round of FISH. This
method ensures thorough removal of the probes (FIG. 9) and permits
subsequent re-probing (FIG. 10).
Example 9--Alignment of Tissue Sections after Multiple Rounds of
Probing and Stripping
[0055] Because DAPI-stained nuclei are typically used to align the
same tissue section across multiple rounds of RNA-FISH, one
limitation to using DNase to strip FISH probes is that the DNA that
binds DAPI is substantially degraded after digesting the first
round of bDNA FISH probes, limiting the application of automated
alignment algorithms for the same tissue sample across multiple
rounds of FISH.
[0056] Multiple rounds of FISH on the same tissue requires
alignment of a reference tissue features across each round (FIG.
11). For this, it was found that, after DNase treatment, DAPI
stains a combination of nuclear and high frequency fibrous
non-nuclear features that are stable across multiple rounds of FISH
(FIG. 12). Using these stable DAPI-binding features, non-linear
registration algorithms can be used to align three dimensional
images of these tissue sections with high precision by combining
three-dimensional affine transformation and non-linear deformation
transformation using, for example the Advanced Normalization Tools
software package (FIG. 12). This algorithm achieves automated
computational alignment of tissue section across multiple rounds of
FISH images over at least four rounds of 3-plex FISH for a total of
12 FISH probes aligned with cellular resolution on a single tissue
sample (FIG. 13).
[0057] It is to be understood that, while the methods and
compositions of matter have been described herein in conjunction
with a number of different aspects, the foregoing description of
the various aspects is intended to illustrate and not limit the
scope of the methods and compositions of matter. Other aspects,
advantages, and modifications are within the scope of the following
claims.
[0058] Disclosed are methods and compositions that can be used for,
can be used in conjunction with, can be used in preparation for, or
are products of the disclosed methods and compositions. These and
other materials are disclosed herein, and it is understood that
combinations, subsets, interactions, groups, etc. of these methods
and compositions are disclosed. That is, while specific reference
to each various individual and collective combinations and
permutations of these compositions and methods may not be
explicitly disclosed, each is specifically contemplated and
described herein. For example, if a particular composition of
matter or a particular method is disclosed and discussed and a
number of compositions or methods are discussed, each and every
combination and permutation of the compositions and the methods are
specifically contemplated unless specifically indicated to the
contrary. Likewise, any subset or combination of these is also
specifically contemplated and disclosed.
* * * * *