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PubMed2000

FISH-microdissection (FISH-MD) analysis of complex chromosome rearrangements.

We combined the techniques of fluorescence in situ hybridization (FISH) and chromosomal microdissection in one experiment (FISH-MD). This novel method permits rapid identification of the composition, origin, and breakpoints of rearranged chromosomes. Rearranged chromosomes are first identified by multicolor-FISH, then the fluorophore-labeled derivative chromosomes are directly isolated by microdissection and reverse painted to identify the breakpoints.

Chromosome Aberrations↗

PubMedDate not supplied

A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.

Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

Actins↗

PubMed2000

Detection of gene amplification in intraductal and infiltrating breast cancer by laser-assisted microdissection and quantitative real-time PCR.

Gene amplification is one essential mechanism leading to oncogene activation which is supposed to play a major role in the pathogenesis of invasive breast cancer. However, using standard methodologies the detection of gene amplifications has been limited especially in small-sized lesions, like pre-invasive precursor lesions. The combination of two novel technologies, laser-based microdissection and quantitative real-time PCR, facilitates the detection of low-level amplifications in morphologically defined lesions. As a model system we investigated in situ breast cancer (ductal carcinoma in situ, DCIS) classified according to the morphology-based Van Nuys grading system for amplification of growth-regulatory genes. In this study 83 formalin-fixed, paraffin-embedded archival DCIS specimens were examined after laser-based microdissection by quantitative real-time PCR using the TaqMan detection system for amplification of the c-erbB2, topoisomerase IIalpha, c-myc and cyclinD1 gene. In a subset of 17 DCIS with adjacent infiltrating tumour components we compared intraductal and invasive tumour components in parallel for differences in amplification status. The combination of these new techniques represents an excellent tool to gain new insights into carcinogenesis by analyzing genetic alterations in morphologically identified heterogeneous lesions in breast cancer progression within the very same specimen or even tissue slide.

Antigens, Neoplasm↗

PubMed2000

Laser-assisted microdissection and short tandem repeat PCR for the investigation of graft chimerism after solid organ transplantation.

The detection of donor-derived cells in the blood and tissues of graft recipients after solid organ transplantation is a readily observed phenomenon called microchimerism. Yet very little is known about the persistence and integration of recipient-derived cells in the transplanted organ, indicating a form of intragraft chimerism. To further study this phenomenon and its possible influence on graft acceptance or rejection, we developed the following novel approach. Immunohistochemically labeled cells were isolated by means of laser-based microdissection and subsequent laser pressure catapulting from paraffine-embedded posttransplantation biopsies. The following use of a highly sensitive PCR assay analyzing one polymorphic short tandem repeat (STR) marker enabled us to clearly identify the genotypes in samples containing as little as 10 isolated cells. The combination of laser-based microdissection and STR-PCR thus provides a powerful tool for the genotyping of even very few cells isolated from routinely processed biopsies after solid organ transplantation.

Cell Separation↗

PubMed2000

Laser microdissection as a new approach to prefertilization genetic diagnosis.

The genetic status of oocytes can be determined by polar body (PB) analysis. Following PB extraction, a genetic evaluation is performed. As each PB contains the complementary genetic material of the oocyte, PB analysis reveals information about its genetic status. Genetically altered oocytes may then be excluded from in vitro fertilization. The aim of our study was to evaluate laser microdissection as a tool for PB extraction purposes. Compared to the PB extraction with a sharp-ending pipette only, we could show that laser microdissection of the zona pellucida (laser zona drilling) with a UV-A laser and subsequent extraction with a blunt-ending pipette decreases the degeneration rate of oocytes. It is shown that laser pressure catapulting of extracted PB enables their contact-free transfer into tubes, thus decreasing the risk of contamination for further analysis.

Cell Separation↗

PubMed2002

Multicolor chromosome banding (MCB) with YAC/BAC-based probes and region-specific microdissection DNA libraries.

Multicolor chromosome banding (MCB) allows the delineation of chromosomal regions with a resolution of a few megabasepairs, i.e., slightly below the size of most visible chromosome bands. Based on the hybridization of overlapping region-specific probe libraries, chromosomal subregions are hybridized with probes that fluoresce in distinct wavelength intervals, so they can be assigned predefined pseudo-colors during the digital imaging and visualization process. The present study demonstrates how MCB patterns can be produced by region-specific microdissection derived (mcd) libraries as well as collections of yeast or bacterial artificial chromosomes (YACs and BACs, respectively). We compared the efficiency of an mcd library based approach with the hybridization of collections of locus-specific probes (LSP) for fluorescent banding of three rather differently sized human chromosomes, i.e., chromosomes 2, 13, and 22. The LSP sets were comprised of 107 probes specific for chromosome 2, 82 probes for chromosome 13, and 31 probes for chromosome 22. The results demonstrated a more homogeneous coverage of chromosomes and thus, more desirable banding patterns using the microdissection library-based MCB. This may be related to the observation that chromosomes are difficult to cover completely with YAC and/or BAC clones as single-color fluorescence in situ hybridization (FISH) experiments showed. Mcd libraries, on the other hand, provide high complexity probes that work well as region-specific paints, but do not readily allow positioning of breakpoints on genetic or physical maps as required for the positional cloning of genes. Thus, combinations of mcd libraries and locus-specific large insert DNA probes appear to be the most efficient tools for high-resolution cytogenetic analyses.

Chromosome Banding↗

PubMed2002

Isolation of a new germ line-specific repetitive DNA family in Acricotopus by microdissection of polytenized germ line-limited chromosome sections from a permanent larval salivary gland preparation.

In Acricotopus lucidus (Diptera, Chironomidae) the germ line-limited chromosomes (Ks) have developed from the soma chromosomes (Ss) by endoreduplication, rearrangements and accumulation of germ line-specific repetitive sequences. For molecular analysis of specific small K sections, microdissection of metaphase Ks generally yields very limited amounts of DNA. In this study, K-specific DNA was microdissected from defined polytenized K sections of X-ray induced K-S-rearrangements of permanent salivary gland chromosome preparations and was then amplified by DOP-PCR. A new germ line-specific tandem repetitive DNA family was isolated by this way from a heterochromatic K segment, characterized and localized on the Ks by FISH. The repetitive elements are related to sequences of earlier described K-specific tandem repetitive DNA families in A. lucidus, but are located mainly in terminal heterochromatin bands of the two largest Ks and only to a limited degree in the paracentromeric K heterochromatin. This demonstrates that a collection of permanent preparations of K-S-rearrangements with polytenized heterochromatic and S-homologous K sections of A. lucidus can be used as a source for obtaining K sequences of defined K parts to investigate the molecular evolution of the Ks.

Animals↗

PubMedDate not supplied

An assessment of MMP and TIMP gene expression in cell lines and stroma - tumour differences in microdissected breast cancer biopsies.

To examine matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) mRNA levels in archival breast cancer biopsies, we employed microdissection to separate tumour tissue from the surrounding breast tissue, or stroma and RT-PCR to determine gross qualitative and small quantitative differences in the patterns of expression. In this study, a significant correlation (p < 0.05, by Mann-Whitney U analysis) between TIMP-2 expression and lymph node involvement was identified, while MMP-11 and TIMP-1 expression patterning also significantly (p < 0.05) differed between those tumours showing calcification and those that did not. When compared by Spearmans' rho correlation analysis, a significant association (p < 0.05, rho = 0.404) was identified in the pattern of MMP-2 and MMP-9 gene expression. In this study, the use of microdissection and a systematic strategy of RT-PCR analysis have allowed us to investigate localized MMP and MMP inhibitor expression within breast tumours. We have identified patterns of gene expression that may further reveal aspects of breast carcinogenesis, and a robust method for examining changes in clinically important genes using archival biopsies and across stroma-tumour boundaries.

Base Sequence↗

PubMed1991

Painting of defined chromosomal regions by in situ suppression hybridization of libraries from laser-microdissected chromosomes.

"Painting" of defined chromosomal regions provides a powerful tool for cytogenetic analyses. Here, we demonstrate that chromosomal in situ suppression (CISS)-hybridization of DNA libraries derived by microcloning laser-microdissected chromosomal regions can be applied to achieve this goal. As an example, we used unbanded metaphase spreads from a female patient carrying a balanced translocation. t(1;7)(1qter----1p36::7q11----7qter). Fragments from the long arms of 130 translocation chromosomes were microdissected. After microcloning, human inserts with an average size of about 3 kb were pooled from 400 recombinant bacteriophage DNA clones and used as a complex probe set in CISS-hybridization experiments. This resulted in painting of the translocation chromosome along the region 7q35 to 1p31. Painted chromosomal subregions in normal chromosomes 1 and 7 were consistent with this finding. This approach may be used to perform painting of any chromosome regions for which microlibraries can be established. Possible applications include the definition of marker chromosomes in clinical and tumor cytogenetics and studies of chromosomal evolution, as well as studies of nuclear chromosome topography in animal and plant species.

Cells, Cultured↗

PubMed1993

Detailed characterization of a human 8q24.1 microdissection library and generation of "sequence-tagged sites".

A total of 533 clones from a human 8q24.1 microdissection library was analyzed by automatic DNA sequencing. Three hundred and thirty-seven different insert sequences were found. The insert size ranged from 45 to 376 bp (mean, 170 bp). Eighty-six percent (291/337) of these sequences were free of repetitive DNA. Each of 19 clones tested was successfully translated into a sequence-tagged site. We conclude that the microdissection library is a rich source of DNA markers for 8q24.1.

Base Sequence↗

PubMed1995

Direct microdissection and microcloning of a translocation breakpoint region, t(1;11)(q42.2;q21), associated with schizophrenia.

We describe the generation of large-fragment microclone libraries from the chromosomal breakpoint of a reciprocal balanced translocation linked to schizophrenia. The abnormality was visible under the phase-contrast microscope, allowing direct dissection from unstained, unbanded metaphases. Two separate microdissection experiments yielded 443 and 672 recombinants, respectively. Following complete EcoRI digestion, inserts with an average size of 0.3 kb (range, 0.2-3 kb) were obtained in the first experiment and 1.5 kb (range, 0.15-6.5 kb) in the second. FISH analysis of pooled clones "painted" back onto the derivative chromosome and assignment of microclones to somatic cell hybrids confirmed the fidelity of the method. Microdissection of chromosome regions identified by karyotype rearrangements in unstained, unbanded metaphases is a potentially powerful tool for positional cloning.

Base Sequence↗

PubMed1996

Generation and mapping of human chromosome 2 microdissection clone-derived STSs.

Twenty-nine new sequence-tagged sites (STSs) were derived from DNA sequences of clones from two human chromosome 2 microdissection libraries. The specificity of the STSs for human chromosome 2 was first demonstrated by PCR amplification of DNA from genomic human and hamster cells and a human chromosome 2-containing human x hamster hybrid cell line. The STSs were then mapped to chromosome 2 by two different approaches. In the first attempt, 12 of the STSs were shown to PCR amplify YAC clones associated with genetic markers on the chromosome. In the second approach, 27 of the STSs were localized to chromosome bands by FISH using cosmid or PAC clones encoding the STSs. The specific STSs mapped to chromosome 2 by these two approaches tie together the genetic and cytogenetic maps of the chromosome at the two termini. The distribution of these STSs further defines the region of the chromosome present in the two microdissection libraries.

Chromosome Banding↗

PubMed1997

Comparative analysis of Y chromosome structure in Bos taurus and B. indicus by FISH using region-specific, microdissected, and locus-specific DNA probes.

Results of fluorescence in situ hybridization (FISH) of Bos taurus and B. indicus Y chromosomes using the bovine locus-specific Y probes BC1.2 and lambda ES6.0 and region-specific probes of B. indicus and B. taurus Y chromosomes, which were generated by microdissection and DOP-PCR, indicate that the Y chromosomes of B. indicus (BIN Y) and B. taurus (BTA Y) differ by a pericentric inversion. Parts of the short and long arms of the Y chromosome in B. taurus and the distal half of the Y chromosome in B. indicus were microdissected, amplified by DOP-PCR, biotinylated, and rehybridized in situ to the corresponding metaphase chromosomes to test the chromosome fragment specificity of the DNA probes. The region-specific painting probes were used for hybridization to metaphase chromosomes of the other species. The DNA painting probes BTA Yp12 and BTA Yq12.1-ter derived from BTA Y hybridized to the distal and proximal halves of BIN Y, respectively. Complex hybridization signals on BTA Yq12.1-->qter were generated with the DNA probe BIN Yqcen-centr (centromere-central) after FISH. The results demonstrate that BTA Yp is homologous to the distal half of BIN Y and that BTA Yq corresponds to the proximal part of BIN Yq. Hybridization of the Y chromosome-specific DNA probes lambda ES6.0 to BTA Yp12-->p11 and near to the telomere of BIN Y and BC1.2 to BTA Yq12-->q13 and to the telomere of BIN Y indicate an opposite orientation of the homologous chromosome fragments BTA Yp and of the distal half of BIN Yq.

Animals↗

PubMedDate not supplied

Detection of heterozygosity loss in microdissected fine needle aspiration specimens of breast carcinoma.

OBJECTIVE: To use the polymerase chain reaction (PCR) to detect loss of heterozygosity (LOH) in microdissected cells form cytologic smears obtained by fine needle aspiration (FNA) from 20 cases of invasive breast carcinoma. STUDY DESIGN: In each case, histologic sections of the primary tumor were also available. Tumor and nontumor cells were dissected from both the cytologic smear and tissue section in all cases except in three smears that showed only tumor cells. RESULTS: LOH was identified in 10 of 19 informative cases using two polymorphic DNA markers at chromosome 11q13 (INT-2, PYGM). The same results were obtained in both the cytologic and histologic specimens, including three cases that had hypocellular cytologic smears. CONCLUSION: FNA of breast lesions provides adequate samples for direct microdissection of the cytologic smear to detect LOH using PCR amplification.

Biopsy, Needle↗

PubMed2003

Gene expression in macrophage-rich inflammatory cell infiltrates in human atherosclerotic lesions as studied by laser microdissection and DNA array: overexpression of HMG-CoA reductase, colony stimulating factor receptors, CD11A/CD18 integrins, and interleukin receptors.

OBJECTIVE: Inflammatory cells play an important role in atherogenesis. However, more information is needed about their gene expression profiles in human lesions. METHODS AND RESULTS: We used laser microdissection (LMD) to isolate macrophage-rich shoulder areas from human lesions. Gene expression profiles in isolated cells were analyzed by cDNA array and compared with expression patterns in normal intima and THP-1 macrophages. Upregulation of 72 genes was detected with LMD and included 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, interferon regulatory factor-5 (IRF-5), colony stimulating factor (CSF) receptors, CD11a/CD18 integrins, interleukin receptors, CD43, calmodulin, nitric oxide synthase (NOS), and extracellular superoxide dismutase (SOD). Several of these changes were also present in PMA-stimulated THP-1 macrophages in vitro. On the other hand, expression of several genes, such as VEGF, tissue factor pathway inhibitor 2, and apolipoproteins C-I and C-II, decreased. CONCLUSIONS: Overexpression of HMG-CoA reductase in macrophage-rich lesion areas may explain some beneficial effects of statins, which can also modulate increased expression of CD11a/CD18 and CD43 found in microdissected cells. We also found increased expression of CSF receptors, IRF-5, and interleukin receptors, which could become useful therapeutic targets for the treatment of atherosclerotic diseases.

Arteriosclerosis↗

PubMed2001

Renal cyclooxygenase-2: evidence for recruitment of thick ascending limb of henle cells in microdissected nephron segments.

Prostaglandins participate in the regulation of sodium and water renal excretion. They are synthesized by cyclooxygenases (COX): the constitutive isoform and the enzyme regulated by physiological stimuli (COX-2). Our previous immunohistochemical studies have demonstrated the presence of COX-2 in a subset of thick ascending limb (TAL) of Henle cells and its induction during the postnatal period and after adrenalectomy. Previous results suggested that this induction phenomenon proceeds by recruitment of TAL cells from the cortex to the outer medulla. The present work aimed to specifically address these preliminary observations by using immunohistochemical techniques in single microdissected nephron segments. Normal adult rats, adrenalectomized rats, adrenalectomized rats on dexamethasone and 5, 10, and 15 days postnatal age were used (Sprague-Dawley rats, n= 5 each group). Glomeruli and different segments of nephron were microdissected from collagenase-treated kidney tissue. Tubules were immunostained with specific antibodies against COX-2. We confirmed that COX-2 was localized exclusively in TAL segments; it was induced after adrenalectomy and during postnatal age, peaking at 15 days after birth. We provided morphological evidence that the induction of COX-2 along TAL proceeded in a defined pattern by recruitment of cells from the cortical portion close to the glomeruli toward the outer medulla. No COX-2 was observed in the post-macula densa portion of the segments. Our results provide the anatomical basis for the contribution of COX-2 in physiological mechanisms such as renin secretion, tubuloglomerular feedback, and the interaction with neuronal NO synthase at the juxtaglomerular apparatus.

Adrenalectomy↗

PubMed1996

Repair of naphthalene-injured microdissected airways in vitro.

Nonciliated bronchiolar epithelial (Clara) cells, as both the primary target for metabolically activated pulmonary toxicants and the progenitor cell for repair after bronchiolar injury, are critical for distal airway epithelial function and regeneration. Previously, we described a model system whereby differentiated Clara cells can be maintained in culture using explants of microdissected distal airways. The purpose of this study is to establish whether distal airway explants can be used to study bronchiolar epithelial repair in vitro. Lungs from adult mice treated with naphthalene, a metabolically activated Clara cell cytotoxicant, or vehicle were inflated with agarose and distal airways were microdissected. Distal airway explants were cultured for up to 7 days in serum-free medium. Clara cells in explants from naphthalene-treated mice exhibited the characteristic cytotoxic responses previously reported in vivo when maintained in vitro: cell swelling, formation of cytoplasmic vacuoles, and exfoliation of injured cells into the airway lumen 1 to 2 days after injury (DAI). Epithelial cells squamated to cover the injured area 2 to 4 DAI. At 7 DAI, the epithelium generally consisted of cuboidal cells. Proliferating cells and marker proteins for differentiated Clara cells (Clara cell 10 kD secretory protein, or CC10, and cytochrome P450 monooxygenase isozyme 2B, or CYP2B) were detected immunochemically and their pattern of distribution during the injury and repair response in vitro paralleled the pattern of cell regeneration seen previously in vivo. We conclude that Clara cells in explants from defined regions of murine tracheobronchial airways can be used to study the early phases of the repair response to naphthalene injury, including differentiation and proliferation, in vitro.

Animals↗

PubMed2006

Gene expression in rat lacrimal gland duct cells collected using laser capture microdissection: evidence for K+ secretion by duct cells.

PURPOSE: To compare gene expression profiles of lacrimal gland duct and acinar cells after laser capture microdissection (LCM) and identify molecular networks related to K+ secretion, testing the hypothesis that duct cells are responsible for high K+ levels in tears. METHODS: Frozen sections of lacrimal glands from five rats were subjected to LCM to isolate pure samples of duct and acinar cells. RNA was extracted, amplified, reverse transcribed, and hybridized to rat cDNA microarrays. Paired arrays from ducts and acini of the five animals were scanned and analyzed with in-house software. Gene expression was confirmed with fluorescent antibodies and confocal microscopy. RESULTS: A list of 10,294 genes expressed in ducts and acini was searched using gene ontologies related to ion transport. From a list of 55 genes that were expressed in ducts, a panel of genes hypothesized to be involved in basolateral-to-apical transport of K+ and Cl- was chosen for validation by immunofluorescence and confocal microscopy. This analysis confirmed translation of the genes of interest and showed that NKCC1, Na+,K+-ATPase and the M3 cholinergic receptor are expressed on the basolateral membrane of duct cells, whereas KCC1, IK(Ca)1, CFTR, and ClC3 are apically localized. CONCLUSIONS: Laser capture microdissection in conjunction with gene expression analysis provides an excellent approach for studying lacrimal gland duct cells about which relatively little is known at the molecular level. As demonstrated in a proposed model, the polarized expression of transporters and channels on lacrimal gland duct membranes is consistent with the hypothesis that duct cells secrete the relatively high K+ in lacrimal fluid.

Animals↗