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PubMed2005

Proteomic analysis of human bladder tissue using SELDI approach following microdissection techniques.

Lysing of a complete biopsy sample results in a mixture of desired and undesired proteins, reflecting the originating cell types. Therefore microdissecting tissue material is mandatory prior to sample lysis and all downstream applications of protein analysis (proteomics). The two most important dissecting methods for bladder tissue specimens are manual microdissection and laser microdissection. Sample transfer can further be separated into manual laser pressure catapulting (LPC) and laser capture microdissection (LCM). One of the possible downstream applications of protein analysis is surface-enhanced laser desorption ionization (SELDI) time-of-flight mass spectrometry. The small quantities of tissue obtained by microdissection are sufficient for use in the SELDI technique.

Humans↗
PubMed2006

[DNA genotyping of oral epithelial cells by laser capture microdissection].

OBJECTIVE: The STR genotypping of trace oral epithelial cells which are microdissected by laser capture microdissection system (LCM) is explored. METHODS: The oral epithelial cells are microdissected using a low-power infrared laser by VERITAS Microdissection Instrument. STR loci of Profiler Plus are detected by multiplex PCR procesures. RESULTS: DNA genotyping of 7-8 oral epithelial cells are succeeded, and DNA genotyping of 3-4 oral epithelial cells are failed. CONCLUSION: It is viable in genotyping of trace oral epithelial cells by Laser Capture Microdissection as a new technology of seperating single cell.

Cell Separation↗
PubMed1996

Chromosome microdissection in leukemia: a powerful tool for the analysis of complex chromosomal rearrangements.

In many human cancers the presence of marker chromosomes or unbalanced translocations prevents complete karyotypic analysis. Chromosome microdissection has become an increasingly important method for assessing chromosome rearrangements. However, most studies using chromosome microdissection have been carried out on established cancer cell lines that provide an unlimited supply of abnormal metaphase cells. We have routinely performed microdissection of as few as three marker chromosome copies from short-term cultures of acute myeloid leukemias, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH) to normal metaphase spreads. Using this type of "reverse chromosome painting," we were able to characterize precisely the chromosomal constitution of each marker chromosome in the samples, confirming the diagnostic usefulness of microdissection in cancer cytogenetics. In addition, in one leukemia with atypical cytological features, microdissection enabled us to detect a novel retinoic acid receptor alpha gene rearrangement.

Acute Disease↗
PubMed2003

Differential gene expression analysis using paraffin-embedded tissues after laser microdissection.

Recent advances in laser microdissection allow for precise removal of pure cell populations from morphologically preserved tissue sections. However, RNA from paraffin-embedded samples is usually degraded during microdissection. The purpose of this study is to determine the optimal fixative for RNA extractions from laser microdissected paraffin-embedded samples. The integrity of RNA was evaluated with the intactness of 18S and 28S ribosomal RNA by electrophoresis and by the length of individual gene transcripts using RT-PCR. The various fixatives were methacarn (a combination of methanol, chloroform, and acetic acid) and several concentrations of ethanol and isopropanol. Methacarn was the optimal fixative for RNA preservation in paraffin-embedded tissues, which included liver, lung, kidney, muscle, and limb. Based on RT-PCR analysis, methacarn fixed samples exhibited the expected RNA sizes for individual genes such as glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and bone-related genes (e.g., alkaline phosphatase and osteonectin). The laser microdissection technique with methacarn fixation was then applied to analyze the differential gene expression between hypertrophic and proliferative chondrocytes in the growth plate of long bone. The expression of type X collagen, a specific gene for hypertrophic chondrocytes, was only observed in hypertrophic chondrocytes, while type II collagen was observed more broadly in the growth plate as anticipated. Thus, combining laser microdissection with methacarn fixation facilitates the examination of differentially expressed genes from various tissues.

Acetic Acid↗
PubMed2005

Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma.

In order to identify new molecular markers for pancreatic intra-epithelial neoplasias (PanINs), the precursor lesions of pancreatic ductal adenocarcinoma, we established a proteomics approach analysing microdissected PanIN cells. Due to the limited amount of proteins available from microdissection, we developed a procedure including fluorescence dye saturation labelling in combination with high resolution two-dimensional gel electrophoresis. With this procedure we were able to analyse proteins extracted from 1000 microdissected cells with a high resolution of up to 2500 protein spots. Using protein lysates from the pancreatic carcinoma tissue as a reference proteome, we were able to successfully identify the proteins. Thus, we could match approximately 2200 protein spots (92%) of the microdissected sample proteome to the reference proteome for protein identification using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and nanoliquid chromatography-electrospray ionisation tandem mass spectrometry after in-gel digestion. The first proteome analysis of microdissected PanIN-2 grades revealed eight differentially expressed proteins. The differential expression of the three actin filament-associated proteins--transgelin, vimentin and MRCL3 as well as actin itself--indicates a relevant role of the actin cytoskeleton during pancreatic tumour progression. Additionally, two members of the annexin family (annexin A2 and A4) implicate a functional contribution of exocytotic and endocytotic pathways at that stage.

Adenocarcinoma↗
PubMed1999

Microassay of 5'-nucleotidase and adenosine deaminase activity in microdissected nephron segments.

The present study describes a new method for microassay of the activity of 5'-nucleotidase (5'-ND) and adenosine deaminase (ADA) in the microdissected nephron segments. The nephron segments including glomeruli, proximal convoluted and straight tubules (PCT and PST), cortical and medullary thick ascending limbs, and cortical and medullary collecting ducts were microdissected. 5'-ND and ADA in the nondenatured lysate of 20-mm microdissected tubules and 20 glomeruli were separated by agarose gel electrophoresis and by isoelectric focusing, respectively. The gels were incubated with specific substrates and staining dyes to exhibit the dephosphorylation by 5'-ND or deamination by ADA. The enzyme activity was estimated by measuring the intensity of the reaction bands on the gels. The 5'-ND activity was detected in all microdissected tubular segments and glomeruli. Among these nephron segments, PCT and PST exhibited the greatest enzyme activity, averaging 1142 and 939 mU/mg tissue protein, respectively. The activity of ADA was also detected in all tubular segments and glomeruli. However, the greatest activity of this enzyme was found in the glomeruli (649.8 mU/mg protein). Using reverse transcriptase-polymerase chain reaction technique, we verified the presence of mRNA of 5'-ND and ADA in all microdissected tubular segments and glomeruli. Based on these results, we conclude that 5'-ND and ADA are present in all nephron segments studied, but the activity of these enzymes is nonuniformly expressed along the nephron. This microassay is a highly specific, sensitive, and reliable method for the segmental analysis of adenosine metabolism in the kidney.

5'-Nucleotidase↗
PubMed1994

Rapid generation of whole chromosome painting probes (WCPs) by chromosome microdissection.

A strategy for rapid construction of whole chromosome painting probes (WCPs) by chromosome microdissection has recently been developed. WCPs were prepared from 20 copies of each target chromosome microdissected from normal human metaphase chromosomes and then directly amplified by PCR using a universal primer. Fifteen WCPs, including chromosomes 1, 3, 6, 7, 9, 12, 13, 14, 15, 17, 19, 20, 21, 22, and X, have been generated using this strategy. The probe complexity and hybridization specificity of these WCPs have been characterized by gel electrophoresis and fluorescence in situ hybridization. Analysis of WCPs constructed by chromosome microdissection indicated that microdissected WCPs invariably provide strong and uniform signal intensity with no cytologically apparent cross-hybridization. To demonstrate the application of WCPs generated from microdissection, we have used these probes to detect complex chromosome rearrangements in a melanoma cell line, UM93-007. Two different translocations involving three chromosomes [t(1;3;13) and t(1;7;13)] have been identified, both of which were undetectable by conventional banding analysis. Further application of these WCPs (including generation of WCPs from mouse and other species) should greatly facilitate the cytogenetic analysis of complex chromosome rearrangements.

Animals↗
PubMed1994

Use of chromosome microdissection, the polymerase chain reaction, and dot blot hybridization to analyze double minute chromosomes.

The potential usefulness of chromosome microdissection, the polymerase chain reaction (PCR), and dot blot hybridization as a quick screening method for determining the genetic composition of double minute chromosomes (DMs) was evaluated. DMs or abnormally banding regions (ABRs) were microdissected from multidrug-resistant hamster cell lines and amplified with PCR using primers specific for the hamster multidrug-resistance (MDR) gene, pgp 1. The microdissected-PCR-amplified products were shown to (a) hybridize to a 32P-labeled pCHP1 probe for the hamster MDR gene by using dot blot or Southern blot analysis and also (b) hybridize back to the chromosome region from which they were originally dissected by using fluorescent in situ hybridization. Microdissected/PCR-amplified DMs were also shown to hybridize to ABRs. When microdissected DMs and ABRs were amplified using hamster specific Alu primers, the resulting material was shown to hybridize with probes for hamster MDR and Alu. These results suggest that the DMs contained in these MDR hamster cell lines contain Alu-like sequences and the chromosome microdissection-PCR-hybridization approach might be used as a quick screening method for identifying genes amplified in DMs and ABRs in cell lines and human tumor samples.

Animals↗
PubMed2001

A new method for histological microdissection utilizing an ultrasonically oscillating needle: demonstrated by differential mRNA expression in human lung carcinoma tissue.

Molecular analysis of microdissected tissue samples is used for analyzing tissue heterogeneity of histological specimens. We have developed a rapid one-step microdissection technique, which was applied for the selective procurement of tissue areas down to a minimum of 10 cell profiles. The special features of our microdissection system consist of an ultrasonically oscillating needle and a piezo-driven micropipette. The validity of this technique is demonstrated in human lung large-cell carcinoma by real-time quantitative reverse transcriptase-polymerase chain reaction assays of vimentin, cyclin D1, and carcinoembryonic antigen after linear RNA amplification. mRNA expression values of microdissected samples scattered around those of bulk tumor tissue and showed differential mRNA expression between samples of tumor parenchyma and supportive stromal cells for vimentin and carcinoembryonic antigen as confirmed by immunohistochemistry. In conclusion, this procedure requires simple equipment, is easily performed, and delivers microdissected tissue samples of oligocellular clusters suitable for further molecular analysis.

Carcinoembryonic Antigen↗
PubMed1999

Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis.

Microdissection of routinely stained or unstained frozen sections has been used successfully to obtain purified cell populations for the analysis of cell-specific gene expression patterns in primary tissues with a complex mixture of cell types. However, the precision and usefulness of microdissection is frequently limited by the difficulty to identify different cell types and structures by morphology alone. We therefore developed a rapid immunostaining procedure for frozen sections followed by laser capture microdissection (LCM) and RNA extraction, which allows targeted mRNA analysis of immunophenotypically defined cell populations. After fixation, frozen sections are immunostained under RNAse-free conditions using a rapid three-step streptavidin-biotin technique, dehydrated and immediately subjected to LCM. RNA is extracted from captured tissue, DNAse I treated, and reverse transcribed. Acetone-, methanol-, or ethanol/acetone-fixed sections give excellent immunostaining after 12 to 25 minutes total processing time. Specificity, precision, and speed of microdissection is markedly increased due to improved identification of desired (or undesired) cell types. The mRNA recovered from immunostained tissue is of high quality. Single-step PCR is able to amplify fragments of more than 600 bp from both housekeeping genes such as beta-actin as well as cell-specific messages such as CD4 or CD19, using cDNA derived from less than 500 immunostained, microdissected cells. Immuno-LCM allows specific mRNA analysis of cell populations isolated according to their immunophenotype or expression of function-related antigens and significantly expands our ability to investigate gene expression in heterogeneous tissues.

Cells↗
PubMed1998

Laser-assisted microdissection of the zona pellucida facilitates polar body biopsy.

OBJECTIVE: To investigate whether polar body biopsy can be performed after laser microdissection of the zona pellucida (ZP). DESIGN: Mouse zygotes were allocated randomly to three groups. The zygotes were subjected to laser microdissection of the ZP and polar body biopsy (group 1), laser microdissection alone (group 2), or no treatment (group 3). SETTING: University-based IVF program. PATIENT(S): Animal study. INTERVENTION(S): A hole was drilled in the ZP of mouse zygotes using a 1.48-micron noncontact diode laser. A microneedle was inserted and the polar body was aspirated. MAIN OUTCOME MEASURE(S): The efficacy of polar body biopsy after laser microdissection of the ZP was evaluated. RESULT(S): The laser diode beam allowed for precise drilling of a 14- to 18-micron hole in the ZP. Polar bodies could be aspirated without damaging the zygote and did not disintegrate during the biopsy. Zygotes developed to blastocysts and underwent the same hatching as control zygotes. Lower hatching rates were observed in untreated zygotes. CONCLUSION(S): Laser microdissection of the ZP with a noncontact laser system facilitates subsequent polar body biopsy. The use of blunt-ended micropipettes greatly reduces the risk of damage to the zygote or the polar body. This procedure makes polar body biopsy more accurate and effective for preimplantation genetic diagnosis.

Animals↗
PubMed2002

Use of laser microdissection in complex tissue.

Concomitant with the rapid development in biomedical knowledge, including the methods of molecular biology and proteomics, and the manufacture of ever more precise optical instruments, powerful lasers, and sophisticated microcomputing hardware and software, laser microdissection systems have emerged which are now entering the field of routine research. Today, several devices are commercially available, congresses devoted to the latest advances in laser microdissection are now held on regular occasions, and the number of publications based on the use of these techniques has risen to over 250. With laser microdissection, histological treatment, such as chemical or immunological fixation and staining, can readily be combined with methods suitable for molecular biology or proteomics. As the optical, technical, and methodological resolution of polymerase chain reaction (PCR) and microdissection increases, genetic and phenotypic studies of biological material are possible even at the level of single cells and subcellular elements. Moreover, questions such as the paracrine interaction of cells within complex tissues, the development of cancer, and the role of single cells in tissue remodeling or development on the microscopic and molecular level can now be addressed precisely at the molecular level. This chapter reviewed the development of laser microdissection platforms, its potential impact on the future of research, and how, in particular, these technologies can be successfully integrated into modern research and routine histopathological studies of complex tissue.

Animals↗
PubMed2000

Molecular strategies to define HLA haplotype loss in microdissected tumor cells.

Loss of heterozygosity (LOH) of chromosome 6p21 is an important mechanism that generates HLA haplotype loss in various human tumors. This mechanism produces non-reversible HLA-deficient tumor cells that can escape T cell immune responses in peptide-vaccinated cancer patients. However, the exact frequency of this mechanism is still unknown, because contaminating stroma in solid tumor tissues masks the tumor DNA obtained from solid samples. A microdissection technique was applied to 4-8 microm sections of cryopreserved tumor tissues from a group of colorectal and laryngeal carcinomas. Fifteen patients were analyzed for the presence of LOH associated with the beta(2)-microglobulin gene in chromosome 15, and five patients for LOH associated with HLA genes in chromosome 6. In two cases, autologous metastasis tissue samples were also available. The patients were selected for showing an altered HLA class I tumor phenotype as determined by immunohistological techniques. DNA was obtained from this microdissected material and amplified in order to detect the presence or absence of nine previously selected microsatellite markers. HLA sequence based typing (SBT) was also applied to these microdissected DNA samples to define the HLA genotype. Microdissection greatly improved the definition of LOH, with nearly 100% signal reduction in one of the alleles. In addition, this procedure allowed us to detect beta(2)-microglobulin LOH in tumors that expressed some HLA molecules. Our data indicate that this procedure can be successfully applied to microdissected samples from solid tumors, thus enhancing the power and sensitivity of LOH detection.

Colorectal Neoplasms↗
PubMed1993

Generation of band-specific painting probes from a single microdissected chromosome.

We have developed a modified strategy for the generation of regional probes for human chromosomes by microdissection and degenerate oligonucleotide primed PCR. This modification dramatically increases the efficiency of amplification by pretreatment of the dissected chromatin with topoisomerase I (Topo I) before PCR. This protocol has enabled us to construct region-specific probes for fluorescence in situ hybridization (FISH) from a single microdissected chromosome. Results are presented which convincingly demonstrate that this new method generates high intensity region-specific FISH probes, while at the same time significantly decreasing the time-consuming and labor-intensive aspects of microdissection. The reduction of the number of copies required to generate a useful probe also significantly decreases the risk of contamination during the microdissection process. We believe this advance will allow microdissection to be more widely used in the cytogenetic analysis of chromosome rearrangements in both cancer and hereditary diseases. In addition, this method now makes it possible to construct a series of non-overlapping band-specific DNA microclone libraries to provide complete coverage of individual chromosomes for physical mapping.

Base Sequence↗
PubMed1997

Comparison of genetic changes in frozen biopsies and microdissected archival material from the same colorectal liver metastases.

Microdissection of tissue sections from formalin-fixed, paraffin-embedded tumor material allows separation of microscopic sites within a sample. DNA can easily be extracted, and polymerase chain reaction (PCR) technology makes it possible to perform different molecular biologic analyses on small cell populations. The presence of normal cells or tumor heterogeneity may cause false negatives in allelic imbalance (AI) studies. Microdissected well-defined cell populations from a tumor section are assumed to increase the sensitivity of AI analyses. The present study has evaluated this in colorectal liver metastases by comparing genotypes in frozen biopsies with genotypes in microdissected archival samples from the same patients. Constitutional genotypes were obtained from corresponding peripheral blood leukocytes as well as normal liver tissue. Archival samples (n = 43) from 16 patients were analyzed after microdissection with 2-5 of 10 selected microsatellite markers. Frozen biopsies from one metastasis of each patient had previously been investigated at numerous microsatellite loci. From those results we selected, for the comparable analysis of archival samples, 41 tumor genotypes at 10 loci representing 11 heterozygotes, 13 AI, 7 losses of heterozygosity (LOHs), 8 homozygotes, and 2 microsatellite unstable cases. The microdissected samples revealed AI or a complete loss of one allele (LOH) in 5 of 11 (45%) genotypes that were previously evaluated as unchanged (retained heterozygosity) in the frozen biopsies, and LOH in 8 of 13 (62%) genotypes at loci known to exhibit AI in the frozen biopsies. Microsatellite instability, LOH, and homozygosity found in the frozen samples were all confirmed by analyses of the archival material. Intertumoral genetic heterogeneity was found in samples from two patients. The same allelic intensities were seen in DNA from tumor-close liver tissue as in blood DNA from the same patient except in one sample. The present study shows a 54% increase in sensitivity of genetic alterations if pure tumor cell components are used (five "new" AIs and LOHs and eight "new" LOHs among previously scored heterozygotes [n = 11] and AI [n = 13], respectively). In total, a 93% success rate (108/115 analyses) was obtained using standard PCR conditions for the 10 selected markers. The fact that standard PCR conditions and 5-micron tumor sections are used shows how easy these analyses are to perform, and that only minor amounts of valuable archival material is used.

Colorectal Neoplasms↗
PubMed1984

Alteration with dietary state of the activity and zonal distribution of adenylate cyclase stimulated by glucagon, fluoride and forskolin in microdissected rat liver tissue.

Adenylate cyclase activated by glucagon, fluoride and forskolin was measured in liver homogenates and microdissected periportal and perivenous tissue of fed and fasted rats. A radiochemical microtest, more sensitive by 2-3 orders of magnitude as compared with the usual assay, was established for the determination of the activity in liver samples corresponding to 200-600 ng dry weight. In liver homogenates from fasted as compared to fed animals the glucagon-stimulated and fluoride-stimulated activity was increased by 1.65-fold, while the basal and the forskolin-stimulated activity remained the same. In microdissected tissue of both fed and fasted animals the activity was stimulated in about 60% of the samples by glucagon, fluoride and forskolin (responsive samples). However, in about 40% of the microdissected tissue samples the activity could not be stimulated by any of the above activators (non-responsive samples). In responsive microdissected tissue of fasted as compared to fed animals, the glucagon-stimulated and fluoride stimulated activity but not the basal and the forskolin-activated activity was increased by 2-3-fold. In responsive microdissected samples of fed animals neither the basal nor the stimulated activities showed a significant periportal to perivenous gradient. In samples of fasted animals, however, a zonal gradient was observed for the glucagon-stimulated activity exhibiting a 1.5-fold higher rate in the perivenous zone.

Adenylyl Cyclases↗
PubMed1996

Chromosome microdissection: a brief overview.

Chromosome microdissection arose as a means of facilitating long range physical mapping of chromosome regions involved in either a genetic or malignant disorder. However, with the rapid development of improved techniques for mapping and sequencing the human genome, microdissection is considered by many investigators to be a cumbersome and time consuming procedure. Nonetheless, based on the impressive number of informative diagnostic DNA markers that are now available as a result of this technology, microdissection still must be considered one of the most rapid and direct methods available for generating new DNA markers from any chromosome region, irrespective of its sequence composition. In addition, it remains an important means to dissect DNA markers from any organism, eukaryotic and prokaryotic, and has resulted in generating disease associated DNA sequences from both human and animal genomes. Recently, microdissection of single cells has emerged as a viable alternative for isolating pure populations of specific cell types, especially tumor cells, which can then be studied without background contamination from any other cellular constituents. This overview will provide a glimpse into the present applications of the microdissection technology, as well as the importance this technology will have for future exploration into the human genome.

Animals↗
PubMedDate not supplied

Gene transcript assay by real-time RT-PCR in epithelial breast cancer cells selected by laser microdissection.

The cell type heterogeneity within clinical cancer tissue samples may affect the accuracy of gene expression analysis. In order to validate our laser microdissection (LMD) method using the Leica AS LMD system (LEICA Microsystems), we compared the mRNA levels of three major genes involved in breast cancer (ERalpha, PR, HER2), measured by means of real-time quantitative RT-PCR, in 5000 microdissected malignant epithelial cells and in corresponding bulk tumor homogenates from 14 patients. We also compared the mRNA level results to protein expression measured by immunohistochemistry (IHC) on the same tumors. For the three genes, significant correlations were found between mRNA results obtained on microdissected cells and IHC. Comparison between IHC and mRNA results obtained on microdissected cells and bulk tumors showed that in all cases microdissection enhanced the sensitivity of assessing target gene transcript levels and was essential for their accurate evaluation in heterogeneous tumors.

Adult↗