Over the past decade, many organisms have been the
subject of large-scale genome projects, and as a result a
tremendous number of gene sequences are now ready
for functional analysis. Knowledge of tissues and cells
that express particular genes is key to understanding
Microarrays and similar high-throughput gene expression
technologies are well established and widely used
to determine the expression of large numbers of genes in parallel. Their cellular resolution
and accuracy of expression
patterns, however, are
limited to the level of tissue
dissection. Nonetheless, they
are excellent filters for the selection of genes that are
suitable for analysis by in situ hybridization.
In situ hybridization is a gene expression technique that
provides spatial detail and allows the detection of very
small numbers of positive cells in an intact tissue context.
The technique is vital for the functional analysis of
genes, and it has been used extensively in biological and
medical research for more than 20 years. To allow the
simultaneous analysis of large numbers of genes by in
situ hybridization, the procedure was automated fairly
Automation of nonradioactive in situ hybridization not
only increases throughput, but also overcomes its major
impediments, i.e., that it is technically challenging, laborintensive,
and prone to human error, by exerting accurate
control of critical parameters such as temperature, pipetting
volume, incubation time, and number of repetitions.
There are only a few instruments for automated in situ
hybridization available on the market. For very small,
permeable tissue samples such as animal eggs, small
arvae, or tiny plant root tips, instruments optimized for
whole-mount in situ hybridization (e.g., Intavis) are suitable.
However, the large majority of tissues are too large
for the whole-mount method and have to be dissected,
sectioned, and attached to microscope slides for further
The Max Planck Institute of Experimental Endocrinology
in Hanover and the Swiss lab automation company
Tecan have jointly developed a new system to process
microscope slides with tissue sections. The slides carrying
tissue sections are assembled into flow-through
chambers and remain there until the end of the procedure.
Capillary forces ensure that the tissue sections are
liquid-covered at all times and protected from damage and desiccation. All solutions are added to the chambers
by the computer-controlled liquid-handling system
and displace the solution of the previous incubation. The
instrument accommodates two thermoracks for the
simultaneous processing of 96 slides.
The results described in this report have been generated
by the group of Professor Gregor Eichele, Hanover.
Examples presented are the expression of calbindin in
the adult mouse brain and of neurotrophic tyrosine
kinase type 2 receptor (Ntrk2) in a 14.5-day-old mouse
embryo. More results of the groups large-scale mouse
gene expression project can be viewed at www.genepaint.
org. The nonradioactive automated in situ
hybridization method and its application have been
described previously [1, 2, 3].
Materials and Methods
Preparation of DIG-labeled RNA probe
The DNA template corresponding to the gene of interest
is produced by polymerase chain reaction (PCR) using a
gene-specific primer pair that also comprises the T3, T7,
or SP6 promoter sequences.
Using 1 g of gel-purified
template, DIG-labeled RNA probe is produced by in vitro
transcription according to the instructions of the DIG
RNA Labeling Kit (Roche Applied Science). The probe
concentration is adjusted to 100 ng/l in hybridization
buffer. DIG RNA probes are stored at -20C until used.
Adult mouse brain or 14.5-day-old embryos are isolated,
placed in an embedding chamber containing O.C.T. 4583
(Tissue-Tek, Sakura) and slowly frozen. Tissues are sectioned
using a Leica CM3050S cryostat to a thickness of
20 m, placed on Super Frost Plus microscope slides.
Sections are fixed for 20 minutes in a solution of
4% paraformaldehyde (PFA, EMS) in phosphatebuffered
saline (PBS), washed, acetylated, and dehydrated
through graded ethanol series. The slides can be
stored in air-tight moisture-protected chambers at -80C
for at least 3 months.
After adjusting to room temperature for several hours,
slides are assembled in the flow-through chambers. To
prepare the tissue sections for hybridization with the
RNA probe, they are submitted to a series of prehybridization
treatments. All required solutions are prepared
and placed in heatable or ambient temperature
reservoirs on the robot platform. The robot performs
each step automatically by pipetting the solutions into
the slide chambers according to the programmed script. A wide variety of scripts can be used, corresponding to
the desired in situ hybridization protocol.
DIG-labeled RNA probes are denatured and placed into
the appropriate positions in the heatable microreaction
vial rack on the instrument platform. A minim
um of 120 l
(optimal are 300 l) probe is added per slide, and
hybridization is carried out at 60C overnight. Within the
range of 10 80C, the temperature varies only by
0.5C in each individual flow-through chamber. The
temperature variation across the whole thermorack is
1.0C. This highly accurate temperature control ensures
consistent and reproducible results within one experiment,
and between different experiments.
After hybridization, stringency washes are carried out at
the desired temperature (typically 60C) to remove
unbound RNA probes. The robot pipettes the preheated
washing solutions (SSC/formamide) from their heatable
reservoirs on the platform into the heated flow-through
chambers, so that there is no loss of temperature or bubble
formation on the slides.
At ambient temperature, several blocking steps are carried
out to reduce nonspecific background. After the
blocking steps, antidigoxigenin antibody is applied to the
slides. Typically, these antibodies consist of Fab fragments
that are linked to alkaline phosphatase, peroxidase,
or another enzyme for colorimetric detection
(Roche Applied Science). An optional signal amplification
step (TSA system, Perkin Elmer Lifesciences) helps
to detect transcripts of weakly expressed genes.
After several washing steps to remove unbound antibody,
the substrate for color reaction (BCIP and NBT,
Roche Applied Science) is applied and slides are incubated
until the desired signal intensity is reached. The
reaction can be timed manually or programmed to a
specific time. The reaction is then stopped and the color
precipitate is fixed for slide mounting and microscopy.
Slides are left to dry overnight an
d coverslipped with
aqueous mounting medium.
For automated nonradioactive in situ hybridization, the
Tecan Freedom EVO robot 150/8 pipetting instrument
(eight liquid-dispensing needles) with GenePaint system
components (Figure 1) is used.
Slides generated by the in situ hybridization robot are
coverslipped and photographed in a compound microscope
Leica DMR microscope, a motorized Mrzhuser
stage that accommodates up to eight slides, a Leica
electronic focusing system, a JVC CCD camera, and a
PC-based controller that drives stage and camera. A
detailed version of the procedure can be obtained from
Using the DIG system for nonradioactive in situ hybridization
in combination with the Tecan pipetting instrument,
Georg Eicheles group has successfully adapted in situ
hybridization to high-throughput and routinely obtains
excellent and consistent results (Figures 2 and 3).
The expression patterns are highly specific and reproducible
with a high signal-to-noise ratio. Using a set of
20 slides representing different tissues or developmental
stages per gene, 50 genes can be analyzed per week
if 200 slide positions are available (Figure 1b, c) .
Summary and Conclusion
The DIG system has been applied successfully to highthroughput,
automated in situ hybridization and gives
excellent and consistent results, allowing the routine
analysis of a large number of probes. By automation of
nonradioactive in situ hybridization and adaptation for
hput, the detailed analysis of spatial expression
patterns of large numbers of genes has become
possible and will soon give a new dimension to functional genomics.
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