RAPID COMMUNICATION |
Correspondence to: Sylvie Dumas, LGN, Bât. CERVI, 5ème étage, Hôpital de la Pitié-Salpêtrière, 83, bd de l'Hôpital, 75013 Paris, France. E-mail: sdumas@infobiogen.fr
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Summary |
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A better understanding of biological phenomena involving modulations of gene expression requires quantitative analysis of the expression of several genes in the same structure. For this purpose, we have developed a novel in situ hybridization method to quantify two different mRNA species in the same tissue section simultaneously. Two probes labeled with radioelements of significantly different energies (3H and 33P or 35S) were used to detect the mRNA species. Radioactive images corresponding to the detected mRNA species were acquired with a Micro Imager, a real-time, high-resolution digital autoradiography system. An algorithm was used to process the data such that the initial radioactive image acquired was filtered into two subimages, each representative of the hybridization result specific to one probe. This novel method allows local discrimination and quantification of the respective contributions of each label to each pixel and can therefore be used for quantitative analysis of two mRNAs with a resolution of 1520 µm. (J Histochem Cytochem 48:15871591, 2000)
Key Words: in situ hybridization, double radioactive labeling, Micro Imager, co-detection, gene expression
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Introduction |
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In situ hybridization (ISH) is now a routine method for detection of genetic material. It is used in a large number of biological fields such as anatomy, cellular biology, and regulation of gene expression (for reviews see
In 1994, we described a new ISH approach based on the direct detection of radioactive emission by using the high resolution of a radio imager to analyze mRNA expression in brain tissue sections (
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Materials and Methods |
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Tissue Preparation
One male adult SpragueDawley rat (Iffa Credo; L'Arbresle, France), weighing between 350 and 400 g, was anesthetized with urethane carbamate (1.5 mg/kg), and placed in a stereotaxic frame for electric stimulation. The animal was sacrified and its brain was extracted and frozen in isopentane at -60C. Coronal sections (20-µm-thick) were cut on a cryostat at -22C. Sections were mounted on Superfrost Plus slides and stored at -80C. All experimental procedures were carried out in accordance with the European Communities Council Directive (24.xi.1986) and with the guidelines of the CNRS and the French Agricultural and Forestry Ministry (decree 87848, license number A91429).
Double Radioactive ISH
Two oligonucleotide probes were used for these experiments. One is complementary to part of the syntaxin 1B sequence (35-mer oligonucleotide sequence 5'-GAT GTG TGG GGA GGG TCC TGG GGA AGA GAA GGG TA-3') and the other to part of the Homer sequence (39-mer oligonucleotide sequence 5'-GGT CAG TTC CAT CTT CTC CTG CGA CTT CTC CTT TGC CAG-3'). Oligonucleotides were synthesized in house on a Beckman Oligo 1000DNA synthesizer. The probes were 3' end-labeled with [35S]-deoxyadenosine triphosphate (Amersham; Orsay, France) or [3H]-deoxycytosine triphosphate (Amersham) in a tailing reaction, using terminal deoxynucleotide transferase (Amersham) according to the manufacturer's instructions. The specific activity after labeling was between 1 x 108 and 3 x 109 cpm/µg for each probe.
Coronal brain sections (20-µm-thick) were postfixed in 4% paraformaldehyde in PBS, then washed three times for 10 min in PBS baths and dried in a 95% ethanol bath immediately before hybridization. The hybridization solution was composed of 50% Amersham in situ hybridization buffer, 40% formamide (Eurobio; Les Ulis, France), 0.1 M dithiothreitol (DTT) (Euromedex; Souffel Weyersheim, France), and 0.5 mg/ml poly(A) (Roche; Saint Quentin Fallavier, France). Both probes were diluted 1:100 in the hybridization solution and 75 µl of the mixture was applied to each brain slice. Sections were incubated overnight at 50C under Fuji parafilm coverslips, then washed twice for 15 min in 1 x standard saline citrate (SSC)/10 mM DTT at 53C, twice for 15 min in 0.5 x SSC/10 mM DTT at 53C, and once in 0.5 x SCC/10 mM DTT at room temperature and then dried in a 95% ethanol bath. Radioactive signals from the sections were acquired with a Micro-Imager (Biospace Mesures; Paris, France), which is a real time, high-resolution digital autoradiography system.
Imaging Equipment for Radiolabeled Tissue Sections
To analyze the double radiolabeling in the sections, a thin foil of scintillating paper is brought into contact with the sections. ß-Particles emitted by the sections are identified through acquisition of the light spot emissions in the scintillating foil by a CCD camera coupled to an image intensifer. The result of the acquisition is displayed live on a computer. During the acquisition, radioactive images can be saved at any time to be analyzed. Acquisition is stopped once the number of disintegrations acquired is statistically sufficient. The filter processing allows discrimination and quantification in each pixel of the respective contributions of the two radioelements of significantly different energies.
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Results |
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To show the feasibility of simultaneous ISH of two radioactive probes on the same section, electric stimulations were used for neuronal activation in one side of a rat brain. The expression of the two genes studied, Homer and syntaxin 1B, which are differentially regulated, was followed. Because this work aimed only to validate the technique, the physiological implications of the findings are not addressed.
The principle of the double-labeling ISH technique is illustrated in Fig 1. [35S]-dATP and [3H]-dCTP were chosen to label two different probes that were simultaneously hybridized to a single tissue section. The Micro Imager was used to acquire the signal from the hybridized section in a single step. The initial image was consequently filtered to segregate the image corresponding to [3H]-ß disintegrations (Fig 2C) from that corresponding to [35S]-ß disintegrations (Fig 2E). The quantitative data for both 3H and 35S labeling were incorporated into a single image (Fig 2A). In this figure, green corresponds to the cells that contain only the mRNA detected by the 3H-labeled probe, red to those that contain only the mRNA detected by the 35S-labeled probe, and shades of yellow to those that contain both.
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To control the filtering segregating [35S]-ß disintegrations from the [3H]-ß-disintegrations, three control dots were spotted by hand on the slide. The dots contained, respectively, the 3H-labeled probe (200 cpm), a mix of the 3H (200 cpm)- and the 35S (200 cpm)-labeled probes, and the 35S-labeled probe (200 cpm). All three spots are observed in the image with both labels (Fig 2A) and only two dots after filtering, as expected (Fig 2C and Fig 2E). Quantification of the radioactivity emitted by each dot before and after filtering gave values in accordance with the amount of radioactivity spotted.
The expression of the two mRNAs along a line drawn on the section is quantitatively analyzed in Fig 2 for illustration. The respective contribution of each label to each pixel along this line is shown on graphs (Fig 2B, Fig 2D, and Fig 2F). From the graphs, cells that differentially expressed the two mRNA species are clearly identified and others expressed them at a similar level. This novel method allows quantitative comparison of the expression of these mRNAs in different cells. For example, the cells indicated by Arrow 4 expressed about five times as much mRNA hybridizing with the 3H-labeled probe as the cells indicated by Arrow 3. They also contain large amounts of mRNA detected by the 35S-labeled probe, whereas the amount in the cells indicated by Arrow 3 is barely detectable (Fig 2).
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Discussion |
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A number of ISH protocols have been developed. They use either enzymatically synthesized RNA and DNA probes or chemically synthesized DNA probes ("oligodeoxynucleotide" probes). Standard protocols use either nonradioactive or radioactively labeled probes. The method of signal detection used depends on the required level of resolution and sensitivity and also on the physiological context (for reviews see
Nonradioactive probes are mainly used for anatomic analyses of gene expression because they provide the greatest spatial resolution and they allow detection of several mRNAs in the same tissue section (peroxidase/alkaline phosphatase) (
In contrast, radioactive labeling allows precise measurement of the level of gene expression (for reviews see
For analysis of the regional distribution of mRNA, storage phosphor screens [resolution of 80 µm (3H) and 180 µm (35S/14C)] and autoradiographic films (2030-µm) allow quantification of signals with exposure times of several days to weeks for films and eightfold less for storage phosphor screens. To detect mRNA in individual cells, the hybridized sections are usually dipped in nuclear emulsion: the amount of the mRNA can be quantified at a cellular level by counting grains. The exposure time required for this technique is often long, from several weeks to several months depending on the amount of mRNA in the tissue (for a review see
Here, we demonstrate that the Micro Imager, in contrast, allows quantitative co-detection. Moreover, this is performed in real time, with a high dynamic range (104), satisfactory resolution (15 µm), and exposure times 10 times shorter than autoradiographic films and 50 times shorter than emulsion (
Our ISH experiments, performed with two different labeled probes (3H/35S), demonstrate the feasibility of double-labeling procedures to study simultaneously the expression of different mRNA species in a single tissue section. To our knowledge, this is the first article reporting ISH detection of more than one transcript and the quantification of their respective expression, allowing the comparison of expression of several genes at the cellular level. The findings with this approach were compared with those obtained by independent single-labeling ISH experiments on adjacent sections. As expected, the expression patterns observed were qualitatively and quantitatively similar.
Two probes hybridized on the same section can be distinguished from each other only if the radioisotopes used to label them have different emission energy spectra. We labeled one probe with 3H and the other with either 33P or 35S. 35S and 33P have similar spectra but different half-lives. However, the 3H energy spectrum is clearly different from those of 33P and 35S (
In situ hybridization has already made an enormous contribution to our understanding of how cellular events interrelate and how mRNA is organized, spliced, and transported. Radioactive detection may now further improve the power of this approach and is suitable for gene expression screening on tissue sections. It may also allow novel types of experiments, e.g., co-detection of an mRNA species (with a radiolabeled nucleotide probe) and a protein (with a 125I-radiolabeled antibody). Furthermore, the co-detection of two radioactively labeled species could be used in conjunction with the detection of other molecules using nonradioactive labeled probes or reagents. This would allow the quantitative and qualitative analysis of five markers on a single tissue section, two of them being labeled with radioactive molecules.
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Acknowledgments |
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Supported by the Centre National de la Recherche Scientifique and the Conseil Régional de l'Ile de France. H. Salin was supported by a PhD studentship from the Ministère de l'Education Nationale de la Recherche et de la Technologie.
Received for publication August 1, 2000; accepted August 2, 2000.
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