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									                                             The DIG System – Nonradioactive Automated
                                             High-Throughput In Situ Hybridization:
                                             a Powerful Tool for Functional Genomics Research
                                             Sabine Hantke
                                             Specialist for In Situ Hybridization and Immunohistochemistry Techniques,
                                             Cologne, Germany
                                             s.hantke@t-online.de



                                              Introduction                                                in parallel. Their cellular reso-
                                                                                                          lution and accuracy of expres-
Molecular Biology




                                              Over the past decade, many organisms have been the          sion patterns, however, are
                                              subject of large-scale genome projects, and as a result a   limited to the level of tissue          Sabine Hantke
                                              tremendous number of gene sequences are now ready           dissection. Nonetheless, they
                                              for functional analysis. Knowledge of tissues and cells     are excellent filters for the selection of genes that are
                                              that express particular genes is key to understanding       suitable for analysis by in situ hybridization.
                                              gene function.
                                                                                                          In situ hybridization is a gene expression technique that
                                              Microarrays and similar high-throughput gene expres-        provides spatial detail and allows the detection of very
                                              sion technologies are well established and widely used      small numbers of positive cells in an intact tissue con-
                                              to determine the expression of large numbers of genes       text. 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
                                           Figure 1 a-c:   a                                              simultaneous analysis of large numbers of genes by in
                      (a) Tecan Freedom EVO robot                                                         situ hybridization, the procedure was automated fairly
                             150/8 platform carrying                                                      recently.
                                the GenePaint system
                      components. Visible are from                                                        Automation of nonradioactive in situ hybridization not
                           left to right: two chamber
                                                                                                          only increases throughput, but also overcomes its major
                             racks, four medium-size
                                                                                                          impediments, i.e., that it is technically challenging, labor-
                        heatable reagent reservoirs,
                        a heatable rack for reaction
                                                                                                          intensive, and prone to human error, by exerting accurate
                              tubes, 15 small reagent                                                     control of critical parameters such as temperature, pipet-
                          reservoirs and three large                                                      ting volume, incubation time, and number of repetitions.
                                                           b
                                    reagent reservoirs.
                     (b) Tecan GenePaint chamber                                                          There are only a few instruments for automated in situ
                     rack with eight pipets located                                                       hybridization available on the market. For very small,
                    above one row of flow-through
                                                                                                          permeable tissue samples such as animal eggs, small
                    chambers. With their backs the
                                                                                                          larvae, or tiny plant root tips, instruments optimized for
                         slides are in direct contact
                      with the heat-exchange wall.
                                                                                                          whole-mount in situ hybridization (e.g., Intavis) are suit-
                          (c) Tecan GenePaint flow-                                                       able. However, the large majority of tissues are too large
                          through chamber carrying                                                        for the whole-mount method and have to be dissected,
                      tissue sections (purple) after                                                      sectioned, and attached to microscope slides for further
                                  in situ hybridization.   c                                              analysis.

                                                                                                          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 carry-
                                                                                                          ing tissue sections are assembled into flow-through
                                                                                                          chambers and remain there until the end of the proce-
                                                                                                          dure. Capillary forces ensure that the tissue sections are
                                                                                                          liquid-covered at all times and protected from damage

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                                                                                            BIOCHEMICA · NO. 1 · 2004
and desiccation. All solutions are added to the cham-       A wide variety of scripts can be used, corresponding to
bers by the computer-controlled liquid-handling system      the desired in situ hybridization protocol.
and displace the solution of the previous incubation. The
instrument accommodates two thermoracks for the             Hybridization
simultaneous processing of 96 slides.                       DIG-labeled RNA probes are denatured and placed into
                                                            the appropriate positions in the heatable microreaction
The results described in this report have been generated    vial rack on the instrument platform. A minimum of 120 µl
by the group of Professor Gregor Eichele, Hanover.          (optimal are 300 µl) probe is added per slide, and
Examples presented are the expression of calbindin in       hybridization is carried out at 60°C overnight. Within the
the adult mouse brain and of neurotrophic tyrosine          range of 10°– 80°C, the temperature varies only by
kinase type 2 receptor (Ntrk2) in a 14.5-day-old mouse      ± 0.5°C in each individual flow-through chamber. The
embryo. More results of the group’s large-scale mouse       temperature variation across the whole thermorack is
gene expression project can be viewed at www.gene-          ±1.0°C. This highly accurate temperature control ensures




                                                                                                                                  Molecular Biology
paint.org. The nonradioactive automated in situ             consistent and reproducible results within one experi-
hybridization method and its application have been          ment, and between different experiments.
described previously [1, 2, 3].
                                                            Posthybridization treatments
Materials and Methods                                       After hybridization, stringency washes are carried out at
                                                            the desired temperature (typically 60°C) to remove
Preparation of DIG-labeled RNA probe                        unbound RNA probes. The robot pipettes the preheated
The DNA template corresponding to the gene of interest      washing solutions (SSC/formamide) from their heatable
is produced by polymerase chain reaction (PCR) using a      reservoirs on the platform into the heated flow-through
gene-specific primer pair that also comprises the T3, T7,   chambers, so that there is no loss of temperature or bub-
or SP6 promoter sequences. Using 1 µg of gel-purified       ble formation on the slides.
template, DIG-labeled RNA probe is produced by in vitro
transcription according to the instructions of the DIG      Antibody-mediated detection
RNA Labeling Kit (Roche Applied Science). The probe         At ambient temperature, several blocking steps are car-
concentration is adjusted to 100 ng/µl in hybridization     ried out to reduce nonspecific background. After the
buffer. DIG RNA probes are stored at -20°C until used.      blocking steps, antidigoxigenin antibody is applied to the
                                                            slides. Typically, these antibodies consist of Fab frag-
Tissue preparation                                          ments that are linked to alkaline phosphatase, peroxi-
Adult mouse brain or 14.5-day-old embryos are isolated,     dase, or another enzyme for colorimetric detection
placed in an embedding chamber containing O.C.T. 4583       (Roche Applied Science). An optional signal amplifica-
(Tissue-Tek, Sakura) and slowly frozen. Tissues are sec-    tion step (TSA system, Perkin Elmer Lifesciences) helps
tioned using a Leica CM3050S cryostat to a thickness of     to detect transcripts of weakly expressed genes.
20 µm, placed on Super Frost Plus microscope slides.

Sections are fixed for 20 minutes in a solution of
4 % paraformaldehyde (PFA, EMS) in phosphate-
buffered saline (PBS), washed, acetylated, and dehydrat-
ed through graded ethanol series. The slides can be
stored in air-tight moisture-protected chambers at -80°C
for at least 3 months.

Prehybridization treatments
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 pre-
hybridization treatments. All required solutions are pre-
pared and placed in heatable or ambient temperature
                                                            Figure 2: Coronal cryostat section through an adult mouse brain
reservoirs on the robot platform. The robot performs
                                                            showing strong expression of Calbindin-28K (a gene encoding a
each step automatically by pipetting the solutions into
                                                            calcium-binding protein) in the Purkinje neurons of the cerebellum.
the slide chambers according to the programmed script.

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                                             BIOCHEMICA · NO. 1 · 2004
                                                                                Results

                                                                                Using the DIG system for nonradioactive in situ hybridiza-
                                                                                tion in combination with the Tecan pipetting instrument,
                                                                                Georg Eichele’s 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 repro-
                                                                                ducible with a high signal-to-noise ratio. Using a set of
                                                                                20 slides representing different tissues or developmen-
                                                                                tal stages per gene, 50 genes can be analyzed per week
                                                                                if 200 slide positions are available (Figure 1b, c) [1].
Molecular Biology




                                                                                Summary and Conclusion

                                                                                The DIG system has been applied successfully to high-
                                                                                throughput, automated in situ hybridization and gives
                                                                                excellent and consistent results, allowing the routine
                                                                                analysis of a large number of probes. By automation of
                    Figure 3: Sagittal section through a 14.5-day-old           nonradioactive in situ hybridization and adaptation for
                    mouse embryo showing the expression of the gene             high-throughput, the detailed analysis of spatial expres-
                    (TrkB) encoding the neurotrophic tyrosine kinase            sion patterns of large numbers of genes has become
                    type 2 receptor (Ntrk2). Note wide-spread but
                                                                                possible and will soon give a new dimension to func-
                    specific expression in brain and spinal chord, in
                                                                                tional genomics.
                    various cranial and axial skeletal structures, and in
                    spinal ganglia (tail region).
                                                                                References
                                                                                1. Herzig U et al. (2001), Novartis Found Symp 239: 129–149.
                                                                                2. Carson J et al. (2002), Curr Opin Neurobiol 12: 562–565.
                                                                                3. Reymond A et al. (2002), Nature 420: 582–586.
                    After several washing steps to remove unbound anti-
                                                                                4. DIG Application Manual for Nonradioactive in situ
                    body, the substrate for color reaction (BCIP and NBT,
                                                                                   hybridization (2002), Roche Applied Science.
                    Roche Applied Science) is applied and slides are incu-
                    bated until the desired signal intensity is reached. The    For more information visit the DIG special interest site
                    reaction can be timed manually or programmed to a           www.roche-applied-science.com/dig
                    specific time. The reaction is then stopped and the color
                    precipitate is fixed for slide mounting and microscopy.      Product                           Pack Size       Cat. No.
                    Slides are left to dry overnight and coverslipped with
                                                                                 DIG RNA Labeling Kit              1 kit          1 175 025
                    aqueous mounting medium.                                     DIG RNA Labeling Mix              40 µl          1 277 073
                                                                                                                   (20 reactions)
                    For automated nonradioactive in situ hybridization, the      DIG Nucleic Acid                  1 kit          1 175 041
                                                                                 Detection Kit
                    Tecan Freedom EVO robot 150/8 pipetting instrument
                                                                                 Anti-Digoxigenin-AP,              150 U         1 093 274
                    (eight liquid-dispensing needles) with GenePaint sys-        Fab fragments                     (200 µl)
                    tem components (Figure 1) is used.                           Blocking Reagent                  50 g          1 096 176
                                                                                 Bovine Serum Albumin              20 mg           711 454
                    Microscopy                                                                                     (1 ml)
                                                                                 NBT/BCIP, Stock Solution          8 ml          1 681 451
                    Slides generated by the in situ hybridization robot are
                                                                                 NBT                               3 ml (300 mg) 1 383 213
                    coverslipped and photographed in a compound micro-           BCIP                              3 ml (150 mg) 1 383 221
                    scope Leica DMR microscope, a motorized Märzhäuser           Proteinase K, recombinant,        100 mg        3 115 879
                    stage that accommodates up to eight slides, a Leica          PCR Grade (lyoph.)
                    electronic focusing system, a JVC CCD camera, and a          tRNA                              100 mg            109 495
                                                                                 SP6 RNA Polymerase                5,000 U         1 487 671
                    PC-based controller that drives stage and camera. A
                                                                                 T7 RNA Polymerase                 5,000 U           881 775
                    detailed version of the procedure can be obtained from
                                                                                 T3 RNA Polymerase                 5,000 U         1 031 171
                    the author.


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                                                                 BIOCHEMICA · NO. 1 · 2004

								
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