( ...)The DIG System Nonradioactive Automated,,,High-Throughput In Situ Hybridization:,,,a Powerful Tool for Functional Genomics Research

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 gene function.

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 recently.

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 larvae, 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 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 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.

Tissue preparation

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.

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 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.

Hybridization

DIG-labeled RNA probes are denatured and placed into the appropriate positions in the heatable microreaction vial rack on the instrument platform. A minimum 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.

Posthybridization treatments

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.

Antibody-mediated detection

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 and 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.

Microscopy

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 the author.

Results

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) [1].



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 high-throughput, 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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