BRIEF REPORT |
Correspondence to: Robert A. Lersch, Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS 74-157, Berkeley, CA 94720. E-mail: ralersch@lbl.gov
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Summary |
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The microarray format of RNA transcript analysis should provide new clues to carcinogenic processes. Because of the complex and heterogeneous nature of most tumor samples, histochemical techniques, particularly RNA fluorescent in situ hybridization (FISH), are required to test the predictions from microarray expression experiments. Here we describe our approach to verify new microarray data by examining RNA expression levels of five to seven different transcripts in a very few cells via FISH. (J Histochem Cytochem 49:925926, 2001)
Key Words: spectral imaging, FISH, microarray, RNA, cancer
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Introduction |
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OUR LABORATORY is adapting microarray technology to identify new genes involved in the formation of tumors in the thyroid, breast, and prostate. Previously, the transcription levels of only a few genes could be assayed per experiment. Microarrays circumvent this limitation. However, a second problem limits progress in cancer research. Most tumors are a mix of cell types, due in part to the normal complexity of the tissue and in part to the complexity of tumors as they evolve from benign to metastatic. If researchers collect microarray data without confronting the problem of tumor heterogeneity, important correlations will be missed.
Preliminary studies performed in our lab and elsewhere indicated that solid tumors are heterogeneous with respect to oncogene expression. Fig 1 shows the expression of brk (
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To address this problem, we are developing a system to simultaneously measure cell-by-cell RNA levels of several different markers. The proposed scheme to discriminate benign and malignant neoplasms and to identify new prognostic markers will take advantage of new methods and our experience with oncogene activation in thyroid tumors. Our short-term goal is to develop a system which determines the relative level of expression of five tyrosine kinase genes using FISH-based methods and Spectral Imaging (SIm). Existing SIm instrumentation can record fluorescence spectra from 400 nm to 1100 nm with about 10-nm resolution, whereas the resolution of a light microscope is about 1 µm.
SIm combines the techniques of fluorescence microscopy, charge-coupled device (CCD) camera, and Fourier spectroscopy. The light emitted from each point of the sample is collected with the microscope objective and sent to a collimating lens. This light travels through an optical head (interferometer), is focused on a CCD camera, and the resulting data are processed with a computer. The interferometer divides each incoming beam (light from the microscope) into two coherent beams and creates a variable optical path difference (OPD) between them. The beams are then combined to interfere with each other. The resulting interference intensity is measured by the CCD detector as a function of the OPD. The intensity vs OPD is an "interferogram." The spectrum, i.e., intensity, as a function of wavelength can be recovered from the interferogram using Fourier transforms. The first published application of SIm (spectral karyotyping or SKY) (450 nm to
850 nm).
Although we can use much of the existing technology, the ratio-labeling color scheme for SKY will not work for RNA detection because different labeled cDNA probes might co-localize. Therefore, we will label each cDNA probe with a unique reporter. The fluorescence spectra of the different reporter molecules can partially overlap, because the signals can be resolved by a computer algorithm termed "Spectral Un-Mixing (SUN)" (Applied Spectral Imaging; Carlsbad, CA). SUN enables us to deconvolute overlapping spectra and recover single component images from the spectral image.
The existing commercial SKY metaphase chromosome analysis software will be modified to increase its automated signal processing, RNA identification in interphase cells, integration of cDNA probe signals, and databasing of results. We will only refine the analysis software; the acquisition methods of capturing a spectral image and a separate high-contrast monochrome DAPI image will remain unchanged. A prototype of this system for analysis of protein markers has already been developed (
Initially we will hybridize cDNA probes to brk, ret, c-met, trk, axl/ufo (obtained from Research Genetics; Huntsville, AL) using five cyanine dyes (Amersham; Arlington Heights, IL). On the basis of previous microarray results, we will also add members of the Eph tyrosine kinase family. cDNA probes will be prepared by incorporating fluorochrome-labeled deoxynucleoside triphosphates by random priming or PCR amplification (
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Footnotes |
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Presented in part at the Joint Meeting of the Histochemical Society and the International Society for Analytical and Molecular Morphology, Santa Fe, NM, February 27, 2001.
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Acknowledgments |
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Supported by a grant from the Director, Office of Science, Office of Biological and Environmental Research, US Department of Energy, under Contract DE-AC03-76SF00098, by the Cancer Research Foundation of America and grants from the Cancer Research Program, US Army Medical Research and Material Command, US Department of the Army (DAMD17-99-1-9250, PC991359).
Received for publication December 4, 2000; accepted February 16, 2001.
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