In situ hybridization is a type of hybridization that uses a labeled
complementary DNA, RNA or modified nucleic acids strand to localize a
specific DNA or RNA sequence in a portion or section of tissue, or, if the
tissue is small enough, in the entire tissue, in cells, and in circulating
tumor cells. This is distinct from immunohistochemistry, which usually
localizes proteins in tissue sections. In situ hybridization is a powerful
technique for identifying specific mRNA species within individual cells in
tissue sections, providing insights into physiological processes and disease
pathogenesis. However, in situ hybridization requires that many steps
be taken with precise optimization for each tissue examined and for each probe
used. In order to preserve the target mRNA within tissues, it is often
required that crosslinking fixatives be used.
In situ hybridization is used to reveal the location of specific nucleic acid
sequences on chromosomes or in tissues, a crucial step for understanding the
organization, regulation, and function of genes. The key techniques currently
in use include: in situ hybridization to mRNA with oligonucleotide and RNA
probes; analysis with light and electron microscopes; whole mount in situ
hybridization; double detection of RNAs and RNA plus protein; and fluorescent in
situ hybridization to detect chromosomal sequences. DNA ISH can be used to
determine the structure of chromosomes. Fluorescent DNA ISH can, for example, be
used in medical diagnostics to assess chromosomal integrity. RNA ISH is used
to measure and localize RNAs within tissue sections, cells, whole mounts,
and circulating tumor cells. In situ hybridization was invented by Joseph G.
Gall and Mary-Lou Pardue. Process
For hybridization histochemistry, sample cells and tissues are usually treated to
fix the target transcripts in place and to increase access of the probe. As
noted above, the probe is either a labeled complementary DNA or, now most
commonly, a complementary RNA. The probe hybridizes to the target sequence at
elevated temperature, and then the excess probe is washed away. Solution
parameters such as temperature, salt, and/or detergent concentration can be
manipulated to remove any non-identical interactions. Then, the probe that was
labeled with either radio-, fluorescent- or antigen-labeled bases is localized
and quantified in the tissue using either autoradiography, fluorescence
microscopy, or immunohistochemistry, respectively. ISH can also use two or
more probes, labeled with radioactivity or the other non-radioactive labels, to
simultaneously detect two or more transcripts.
An alternative technology, branched DNA assay, can be used for RNA in situ
hybridization assays with single molecule sensitivity without the use of
radioactivity. This approach can be used to visualize up to four targets in one
assay, and it uses patented probe design and bDNA signal amplification to
generate sensitive and specific signals. Samples are fixed, then treated to allow
RNA target accessibility. Target-specific probes hybridize to each
target RNA. Subsequent signal amplification is predicated on specific
hybridization of adjacent probes. A typical target-specific probe will
contain 40 oligonucleotides, resulting in 20 oligo pairs that bind side-by-side
on the target for detection of mRNA and lncRNA, and 2 oligos or a single pair
for miRNA detection. Signal amplification is achieved via a series
of sequential hybridization steps. A pre-amplifier molecule hybridizes to
each oligo pair on the target-specific RNA, then multiple amplifier molecules
hybridize to each pre-amplifier. Next, multiple label probe oligonucleotides
hybridize to each amplifier molecule. A fully assembled signal amplification
structure “Tree” has 400 binding sites for the label probes. When all
target-specific probes bind to the target mRNA transcript, an 8,000 fold
signal amplification occurs for that one transcript. Separate but compatible
signal amplification systems enable the multiplex assays. The signal can be
visualized using a fluorescence or brightfield microscope.
Basic steps for digoxigenin-labeled probes
permeabilisation of cells with proteinase K to open cell membranes
binding of mRNAs to marked RNA probe antibody-phosphatase binding to
RNA-probe staining of antibody
The protocol takes around 2–3 days and takes some time to set up. Some
companies sell robots to automate the process. As a result, large-scale
screenings have been conducted in laboratories on thousands of genes. The
results can usually be accessed via websites.
See also Chromogenic in situ hybridization
References Jin, L; Lloyd, RV. "In situ
hybridization: methods and applications". Journal of clinical
laboratory analysis 11: 2–9. 002/(SICI)1098-2825(1997)11:13.0.CO;2-F.
PMID 9021518. Comprehensive and annotated in situ
hybridization histochemistry RNA sequencing of pancreatic circulating
tumour cells implicates WNT signalling in metastasis
The Local Transcriptome in the Synaptic Neuropil Revealed by Deep Sequencing and
High-Resolution Imaging External links
In Situ Hybridization at the US National Library of Medicine Medical Subject
Headings In Situ Hybridization of RNA and miRNA
Probes to cells, CTCs, and tissues Whole-Mount In Situ Hybridization of RNA
Probes to Plant Tissues Preparation of Complex DNA Probe Sets
for 3D FISH with up to Six Different Fluorochromes
Transcript In Situ Hybridization of Whole-Mount Embryos for Phenotype
Analysis of RNAi-Treated Drosophila in-situ databases:
Ghost, C. intestinalis transcription factors
Zebrafish gene expression search Mouse MGI GXD gene expression
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