Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
2001-06-13
2004-09-14
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C435S007100, C435S040520, C436S164000, C436S172000
Reexamination Certificate
active
06790636
ABSTRACT:
FIELD OF THE DISCLOSURE
The disclosure relates to methods for rapidly identifying and isolating cells and tissue structures in a manner that preserves biological molecules.
BACKGROUND
Laser Capture Microdissection (LCM) is a recently developed method that enables researchers to conveniently perform microscopic dissections of biological tissue samples and obtain pure populations of defined tissue structures and cells (see for example, Emmert-Buck et al.,
Science
, 274: 998-1001, 1996). LCM combines direct microscopic visualization and a rapid one-step isolation of selected cell populations from tissue sections. In the method, a transparent thermoplastic film (the capture film) is placed over the surface of a tissue section and irradiated with laser pulses that thermally fuse the film to the tissue section. By viewing the tissue section through a microscope, a researcher can direct the laser pulses onto specific portions of the tissue section and “capture” desired cells or tissue structures. Because of the small diameter of the focussed laser beam, small cell clusters or even single cells may be captured. When the overlying film is lifted from the tissue section, the specific portions of tissue that were fused to the film are excised from the tissue section, leaving behind unselected, surrounding tissue. The excised tissue portions may then be removed from the film and utilized, for example, in studies of cell-specific gene expression. The LCM process is shown schematically in FIG.
1
. LCM is especially useful for isolating structures from injured or fibrotic tissue, since manual dissection under these conditions is impossible.
The principal difficulty associated with isolating cells and tissue structures by LCM and other microdissection techniques, however, lies in identification of specific types of cells and specific types of structure within a tissue sample. Cell morphology and histochemical staining methods are adequate to identify cells in certain tissue types, but in heterogeneous tissues, such as those found in the kidney and in many tumors, cells and tissue structures may be morphologically and histochemically indistinguishable. Visual differentiation of cells or tissue structures is especially difficult when tissue samples are prepared as frozen sections and viewed through the capture film.
Immunohistochemical staining is another method for identifying individual cell types and tissue structures within a tissue section that distinguishes cells according to their production of specific antigens (i.e., according to their immunophenotype). Such staining allows morphologically similar but functionally different cells to be differentiated. Unfortunately, immunohistochemical staining regimens typically foster loss of biological molecules, such as mRNA. For example, Jin et al. (Jin et al.,
Lab. Invest
., 79: 511-513, 1999) noted a lower yield of reverse-transcription polymerase chain reaction (RT-PCR) products (i.e., a lower initial mRNA concentration) from tissue samples that were immunohistochemically stained in a conventional manner.
Fend et al. (Fend et al.,
American Journal of Pathology
, 154: 61-66, 1999) developed a rapid immunohistochemical method in which the total time tissue is exposed to aqueous solutions is about 8 minutes. Nonetheless, despite the rapid staining protocol, much mRNA is lost. For example, 99% of the &bgr;-actin mRNA from LCM isolated nephron segments is lost with this staining technique (Kohda et al.,
Kidney International
, 57: 321-331, 2000).
Fink et al. (Fink et al.,
Lab. Invest
., 80: 327-333, 2000) describe immunohistochemical and immunofluorescence methods for identifying cells for laser-assisted microdissection (a technique where a laser beam is used to cut selected portions from a tissue section). The study examined the influence of fixation method, antibodies and staining reagents, incubation and total processing times and digestion with proteinase K on mRNA recovery from microdissected tissue samples. Fink et al. disclose an immunofluorescence method that includes as little as six minutes of exposure of a tissue section to aqueous antibody solutions. Moreover, their results indicated that antibodies themselves contribute to loss of mRNA, and they suggest reducing the number of antibodies as part of an immunofluorescence staining regimen designed to preserve mRNA in microdissected tissue samples. Furthermore, they note that their method for combined immunofluorescent staining and laser-assisted microdissection requires an inconvenient shift from fluorescence imaging to brightfield imaging to perform the actual dissection.
What is needed is a more convenient microdissection method that permits identification of particular cell types and tissue structures through specific binding interactions, such as immunochemical interactions, yet which better preserves biological molecules. A method that preserves mRNA and yet allows identification and dissection of tissues according to their specific binding interactions with other molecules is especially needed to study gene expression at the individual cell or tissue structure level.
SUMMARY
The disclosure provides methods for microdissecting tissue, or preparing tissue for microdissection, by labeling a sample of the tissue by exposing the tissue to a sufficient concentration of a labeled specific binding agent for a sufficiently short period of time to reduce a binding time of the agent to the tissue. These methods allow reduction in the loss of a biomolecule (such as mRNA) in the tissue, while allowing a component of interest in the sample to be detected by a labeled specific binding agent Alternatively, a solvent for the specific binding agent can be selected which avoids or diminishes loss of the biomolecule of interest from the tissue. For example, a non-aqueous solvent could be used, optionally in combination with the reduced binding time.
In particular embodiments the labeled specific binding agent is a fluorescent specific binding agent, and the component of interest in the sample is identified by detecting fluorescence in the tissue, for example fluorescence of a sub-population of cells. Once these components have been identified, they may be microdissected and isolated from other components of the tissue. This approach allows certain subsets of microscopic structures (such as cells that express surface antigens, for example antigens associated with disease) to be microdissected from the tissue, and compared to cells that are not labeled. The preservation of biomolecules in the tissue enhances molecular analysis of the component of interest, for example by permitting more accurate levels of mRNA in the dissected cells to be determined, as a measure of differential gene expression.
Mere mention of an aspect of a method, composition, or device in this Summary is not intended to imply that it is an essential aspect of the invention.
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Taylor et al. (Immunohistochemistry: Principles and Practice, American Society for Microbiology, 1996, pp. 369-379).*
Fend et al., “Immuno-LCM: Laser Capture Microdissection of Immunostained Frozen Sections for mRNA Analysis,”Am. J. Pathol. 154(1):61-66, Jan. 1999.
Goldsworthy et al., “Effects of Fixation on RNA Extraction and Amplication from Laser Capture Microdissected Tissue,”Mol. Carcin. 25:86-91, 1999.
Kohda et al., “Analysis of segmental renal gene expression by laser capture microdissection,”Kidney Int. 57:321-331, 2000.
Darling et al., “Revertant mosaicism: partial correction of a germ-line mutation in COL17A1 by a frame-restoring mutation,”J. Clin. Invest. 103(10):1371-1377, May 1999.
Emmert-Buck et al., “Laser Capture Microdissection,”Science274(5289):998-1001, Nov. 8, 1996.
Fink et al., “Immunostaining and Laser-Assisted Cell Picking fo
Liotta Lance A.
Murakami Hiroshi
Spring Kenneth R.
Star Robert A.
Counts Gary W.
Klarquist & Sparkman, LLP
Le Long V.
The United States of America as represented by the Department of
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