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PubMed2004

FAST-FISH with laser beam microdissected DOP-PCR probe distinguishes the sex chromosomes of Silene latifolia.

We present an improved FISH strategy for differentiating the sex chromosomes of the dioecious model plant, Silene latifolia. Fixed mitotic protoplasts were dropped on a polyethylene naphthalate membrane, the X or Y chromosomes were isolated using nitrogen laser beam microdissection, catapulted by laser pressure, and amplified by DOP-PCR. A modified FAST-FISH protocol based on a short hybridization time combined with a low concentration of probe was used. The success of this approach is demonstrated by the differential labeling of the X and Y chromosomes and it could represent a quick method for comparing organization of plant genomes.

Carbocyanines↗
PubMed2004

Targeted generation of 16 sequence-tagged sites for bovine chromosome region 5q21-q25 by microdissection.

The cattle chromosome region BTA5q21-q25 is associated with immune defense and is linked with several economically important traits for growth, ovulation, twinning and milk yield. Chromosome microdissection of the region was performed to generate fragment-specific DNA libraries and to provide new bovine-specific DNA sequences as starting material for physical fine-mapping approaches or linkage studies. A first library screening resulted in the identification of 16 region-specific sequence-tagged sites (STS). Fifteen STS represent new unique cattle DNA sequences, of which one identifies a new microsatellite. Homology with the gene CDK2 was found for the remaining STS.

Animals↗
PubMed2004

Generation of chromosome painting probes from single chromosomes by laser microdissection and linker-adaptor PCR.

Fluorescence in situ hybridization (FISH) plays an essential role in research and clinical diagnostics. The versatility and resolution of FISH depends critically on the probe set used. Here, we describe an improved approach for the generation of specific DNA probes from single copies of chromosomes. Single chromosomes or single chromosomal regions were microdissected by laser pressure catapulting and amplified using linker-adaptor PCR. The probes were labeled and tested in various scenarios including multicolor-FISH experiments employing up to seven different fluorochromes. FISH confirmed the specific and even staining of the respective chromosomal regions. Furthermore, the capability of these probes to detect even small translocations (<3 Mb) suggests that the dissected regions are completely represented in the generated painting probes.

Chromosome Painting↗
PubMed2003

Laser capture microdissection and advanced molecular analysis of human breast cancer.

Advances in comprehensive genomic and proteomic technologies are providing researchers with an unprecedented opportunity for high-throughput molecular analysis of human breast cancer. Adaptation of these technologies to laser capture microdissection (LCM) is poised to exert dramatic change on the pace of breast cancer research. Although technical limitations have impeded the coupling of these high-throughput technologies to LCM, recent advances have allowed for the successful application of this cellular-based approach to breast cancer, and the results of such studies have provided researchers with unique insight into the disease. This approach holds great potential for rapid advancement in our understanding of breast cancer, and it is hoped that such advancements will lead to novel predictive and therapeutic strategies for women with the disease. This review outlines the current status of the adaptation of advanced molecular technologies to LCM and highlights recent studies in which this approach has been applied to human breast cancer.

Breast Neoplasms↗
PubMed2003

Improved detection of clonality in cutaneous T-cell lymphomas using laser capture microdissection.

BACKGROUND: The diagnosis of cutaneous T-cell lymphoma is a challenge for both the pathologist and the clinician. This is particularly true for distinguishing early-stage mycosis fungoides from dermatitis. In this clinical setting, the presence of a clonal T-cell population supports lymphoma. METHODS: Usually, routinely processed paraffin-embedded material is available for gene rearrangement analysis, and polymerase chain reaction (PCR)-based methods to assess clonality can be performed. One drawback of this approach is that sensitivity is suboptimal in biopsy specimens in which the lymphocytic infiltrate represents only a small percentage of all cells present. Another drawback is that DNA extraction from routinely processed, paraffin-embedded tissue is a time-consuming and labor-intensive procedure which can take up to 5 days in our laboratory. To bypass these problems, we used laser capture microdissection (LCM) to obtain lymphocytic infiltrates from tissue sections of formalin-fixed, paraffin-embedded skin biopsy specimens. This approach allows for more specific PCR assessment of the lymphocytic infiltrate and for rapid DNA extraction and PCR analysis. RESULTS: Using the LCM approach, we could demonstrate clonal T-cell receptor gamma gene rearrangements in biopsy specimens that did not show clonality using DNA extracted by conventional methods from full tissue sections. In addition, DNA extraction and PCR analysis can be performed in 11 h. CONCLUSION: In conclusion, applying LCM to clonality analysis of cutaneous lymphocytic infiltrates is rapid and more sensitive than conventional methods, and we recommend introducing this approach into the routine diagnostic setting.

Clone Cells↗
PubMed2004

In situ-RT and immunolaser microdissection for mRNA analysis of individual cells isolated from epilepsy-associated glioneuronal tumors.

Analysis of gene transcription patterns in complex tissues with multiple cell types is a major challenge. Examination of cellular subpopulations for molecular expression patterns requires their isolation from other surrounding cells. We performed single-cell mRNA analysis to study gangliogliomas obtained from patients with pharmacoresistant epilepsy (n = 6), in order to characterize CD34 expressing cells found in these tumors. Fresh-frozen biopsy tissue was analyzed by initial in situ-reverse transcription (in situ-RT) with oligonucleotides, subsequent immunohistochemistry (IHC) to identify specific cell types, and laser-capture microdissection (LCM, herein termed immuno-LCM) to obtain antigen-expressing cell subpopulations. Isolated complementary DNAs (cDNAs) were then quantified by real time-polymerase chain reaction (RT-PCR). We found that short- vs long-term incubation time for the IHC step did not adversely affect cDNA abundance obtained by subsequent RT-PCR, either for high-abundance (glyceraldehyde dehydrogenase; GAPDH), medium-abundance (glial fibrillary acidic protein; GFAP), or low abundance (neurofilament; NFM) gene transcripts. We also determined that the cellular specificity of capture was excellent, as determined by lack of contamination between different immuno-LCM cell isolates. We were therefore able to examine the lineage expression markers of isolated CD34-expressing cells. We observed coexpression of CD34 and NFM, suggesting neuronal differentiation of the CD34 expressing cellular elements in gangliogliomas. Expression markers for other cellular types (myelin basic protein for oligodendroglia; GFAP for astrocytes) were negative. Our findings support the hypothesis that gangiogliomas contain neuronal elements with compromised or atypical differentiation. We consider that this in situ-RT/immuno-LCM protocol is of general applicability, whereby virtually any primary antibody can be used to facilitate capture of individual cells in tissue sections for molecular analysis.

Adolescent↗
PubMed2004

Absence of Mycobacterium avium subsp. paratuberculosis in the microdissected granulomas of Crohn's disease.

The etiology of Crohn's disease remains unknown with inflammatory, infectious, and/or genetic causes suspected. Granulomatous inflammation is a characteristic feature of the disorder, resembling the tissue response to mycobacterium. Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent in Johne's disease, a chronic ulcerative intestinal condition in cattle, and has been implicated as a likely candidate. We carefully microdissected the granulomas from the paraffin-embedded resection specimens of 18 patients with well-established Crohn's disease. The DNA obtained was PCR amplified for the IS900 and IS1311 repeat elements of MAP, PCR product size maintained at 101 and 124 base pairs, respectively. Archival tissue from bovine Johne's disease was used as a positive control. MAP-specific DNA, confirmed by sequencing and comparison with prototype strain sequence, was appropriately amplified from the positive control. None of the Crohn's disease cases yielded a positive amplification product, failing to support a role for the organism in the pathogenesis of this illness.

Adolescent↗
PubMed2004

Evaluation of p53 mutations in premalignant esophageal lesions and esophageal adenocarcinoma using laser capture microdissection.

p53 mutations have been implicated in the development of esophageal malignancies. The purpose of this study was to assess more accurately the incidence and types of p53 mutations in Barrett's esophagus (BE) with and without dysplasia and in esophageal adenocarcinoma, using pure preparations of epithelial cells obtained by laser capture microdissection (LCM). Assays were performed on paraffin-embedded tissue samples of normal antrum and premalignant and malignant esophageal samples from 57 patients, including 16 controls, 10 with BE metaplasia alone, 20 with BE-associated dysplasia, and 11 with BE-associated adenocarcinoma. All tissues were processed for LCM. DNA was extracted from isolated cells, and polymerase chain reaction (PCR) was performed using oligonucleutide primers for exons 5-8 of p53. PCR products were processed for DNA sequencing. p53 sequence abnormalities were identified in 2/16 cases of normal antrum and regenerative/chemical gastritis, 1/10 cases of BE, 1/20 cases of BE with dysplasia, and 2/11 cases of adenocarcinomas. The abnormalities occurred in exons 7 and 8 in the form of point mutations. Our results, using LCM, show that p53 gene mutations are relatively rare in esophageal preneoplastic and neoplastic conditions. Only point mutations were detected, but no deletions/insertions were identified.

Adenocarcinoma↗
PubMed2005

Does HPV play a role in the etiopathogenesis of ameloblastoma? An immunohistochemical, in situ hybridization and polymerase chain reaction study of 18 cases using laser capture microdissection.

Ameloblastomas are epithelial tumors of odontogenic origin, biologically characterized by local recurrence. Among different etiologic factors, HPV infection has been recently postulated to be somehow involved in ameloblastoma etiopathogenesis. To address this issue, we studied 18 ameloblastomas by means of immunohistochemistry, in situ hybridization (conventional and amplified), polymerase chain reaction and nested-polymerase chain reaction analyses using laser capture microdissection in order to detect the occurrence of HPV in this setting. No evidence of HPV infection was detected by morphological examination, immunohistochemistry, in situ hybridization and conventional polymerase chain reaction, while nested-polymerase chain reaction showed a weak positive band in two cases. However, the subsequent restriction enzyme analysis carried out from the nested-polymerase chain reaction amplification products of these two samples excluded the presence of HPV subtypes 16, 18, 31, 33, 35, 52, and 58. The search for HPV 6 and 11 in the same specimens was also negative. In conclusion, our data do not support an etiopathogenetic evidence for HPV in ameloblastoma.

Adolescent↗
PubMed2003

Detection of deleted mitochondrial DNA in Kearns-Sayre syndrome using laser capture microdissection.

A novel 4949-base pair mitochondrial DNA (mtDNA) deletion was detected in various tissues in a postmortem study of a patient with Kearns-Sayre syndrome (KSS). Deleted mtDNA levels were higher in skeletal muscle and brain and lower in kidney, working myocardium, and endocrine tissues (thyroid, parathyroids, pancreas, and adrenal glands). The distribution of the deletion in skeletal muscle and conducting myocardium was analyzed by means of laser capture microdissection (LCM). In skeletal muscle, the abundance of deleted mtDNA was slightly higher in cytochrome c oxidase (COX)-negative fibers (70%) than in COX-positive fibers (64%), whereas in the conducting myocardium it was lower in the atrioventricular node (9%) than in the sinus node and bundle of His (30% and 32%, respectively). In this study, LCM proved to be a reliable technique for a more accurate assessment of genotype/phenotype correlation when investigating mtDNA-related disorders.

Adult↗
PubMedDate not supplied

Microarray analysis using amplified mRNA from laser capture microdissection of microscopic hepatocellular precancerous lesions and frozen hepatocellular carcinomas reveals unique and consistent gene expression profiles.

The indirect labeling cDNA microarray technique was used to evaluate gene expression profiles of pure cell populations from frozen sections of carcinomas and adenomas harvested from precancerous hepatocellular lesions by using laser capture microdissection (LCM). The levels of differentially expressed genes were investigated using a cDNA microarray with 9,984 features with only 2 ug of two-round amplified aRNA, equivalent to 35 cells from LCM-adenomas and frozen samples of carcinomas from simian virus 40 (SV40) large T antigen transgenic rats. A total of 855 genes were identified as being 3-fold or more differentially expressed in carcinomas or adenomas as compared to normal tissue controls. Among these 855 genes, 71 genes were differentially expressed in both carcinomas and adenomas. Commonly up-regulated genes in both carcinoma and adenomas were 28 while 41 of the 71 genes were commonly down-regulated. Two genes, Igh1 (immunoglobulin heavy chain 1(Serum IgG2a), Image clone ID: 875880) and EST clone (AI893585, Image clone ID: 596604) were more than 7-fold up-regulated in carcinomas and 6-fold down-regulated in adenomas. In Cy5 and Cy3 reciprocal experiments for screening out false positive signals, the amplified carcinomas showed higher Pearson Correlation Coefficient values (-0.94 and -0.92) than the LCM-amplified adenoma samples (-0.79 and -0.84). LCM-amplified samples provided higher signal intensities over backgrounds and a greater average of Cy5:Cy3 ratios. Expression levels of mRNAs from selected genes, determined by using traditional dot blot analysis, revealed that 36 of 40 tested expression profiles were consistent with the microarray data. Thus, amplified aRNA harvested from homogeneous cell types using LCM can be applied to study gene expression profiles by use of microarray analysis.

Animals↗
PubMed2004

DNA microarray analysis of gene expression profiles in deep endometriosis using laser capture microdissection.

Endometriosis, a common gynecological disorder that causes infertility and pelvic pain, is defined as the presence of endometrial glands and stroma within extra-uterine sites. However, despite extensive studies its etiology and pathogenesis are not completely understood. Differentially expressed genes were investigated in epithelial and stromal cells from deep endometriosis and matched eutopic endometrium using cDNA microarrays and laser capture microdissection. Validation of results of several up- and down-regulated genes was performed by quantitative real-time RT-PCR. Our data showed that platelet-derived growth factor receptor alpha (PDGFRA), protein kinase C beta1 (PKC beta1) and janus kinase 1 (JAK1) were upregulated, and Sprouty2 and mitogen-activated protein kinase kinase 7 (MKK7) were downregulated in endometriosis stromal cells, suggesting the involvement of the RAS/RAF/MAPK signaling pathway through PDGFRA in endometriosis pathophysiology. In addition, two potential negative regulators of aromatase expression, chicken ovalbumin upstream promoter transcription factor 2 (COUP-TF2) and prostaglandin E2 receptor subtype EP3 (PGE2EP3), were downregulated in endometriosis epithelial cells, which might result in increased local production of estrogen in endometriosis epithelial cells. Furthermore, three potential candidate genes that might be involved in endometriosis related pain were identified: tyrosine kinase receptor B (TRkB) in endometriosis epithelial cells, and serotonin transporter (5HTT) and mu opioid receptor (MOR) in endometriosis stromal cells were all upregulated. One of the candidate genes, MOR, may be involved in a defective immune system in endometriosis. This study has provided new insights into endometriosis pathophysiology.

Endometriosis↗
PubMed2004

Osteoclast-type giant cell neoplasm of salivary gland. A microdissection-based comparative genotyping assay and literature review: extraskeletal "giant cell tumor of bone" or osteoclast-type giant cell "carcinoma"?

Primary salivary gland tumors resembling giant cell tumor of bone are very rare and have unsettled histogenesis. Both mesenchymal and epithelial origins have been suggested. We review 14 cases in the English-language literature and report another case, the first of which to be studied by microdissection-based microsatellite analysis. One-half of the tumors have been associated with a carcinoma, usually salivary duct carcinoma and carcinoma ex pleomorphic adenoma. Significant differences between this tumor and giant cell tumor of bone were observed. Unlike giant cell tumor of bone, in which the nuclei of the mononuclear and giant cells are similar, those of salivary gland show obvious differences between the nuclei of mononuclear cells and osteoclastic giant cells. In addition and in contrast to giant cell tumor of bone, the mononuclear cells of giant cell tumor of salivary gland express epithelial markers (epithelial membrane antigen, EMA; carcinoembryonic antigen, CEA) and androgen receptor. Genotypically, the microsatellite pattern of the giant cell component is more akin to the carcinomatous component and does not resemble giant cell tumor of bone. Biologically, giant cell tumor of salivary gland tends to be more aggressive than giant cell tumor of bone. We conclude that giant cell tumor of salivary gland is an unusual carcinoma that is not related to giant cell tumor of bone.

Alleles↗
PubMed2003

Detection of fusion gene transcripts in fresh-frozen and formalin-fixed paraffin-embedded tissue sections of soft-tissue sarcomas after laser capture microdissection and rt-PCR.

The diagnosis of small round cell sarcomas is often very difficult, especially when only small biopsy specimens are available for examination. Recent studies have shown that some sarcomas have specific recurrent chromosomal translocations producing chimeric gene fusions, which can be detected by reverse transcription-polymerase chain reaction (RT-PCR), fluorescent in situ hybridization (FISH), or cytogenetic analysis. In this study, 12 cases of well-defined sarcomas including Ewings sarcoma/primitive neuroectodermal tumors (ES/PNET), synovial sarcoma (SS), alveolar rhabdomyosarcoma (ARMS), and desmoplastic small round cell tumors (DSRCT) were used to collect specific numbers of cells by laser capture microdissection (LCM), subsequently used for RT-PCR to detect specific chimeric gene transcripts. Tumor cells from fresh-frozen (FS) tissue sections and paraffin-embedded (PS) tissue sections from the same cases were compared directly to evaluate the sensitivity of FS and PS sections as the starting material for analysis. Samples were used for RNA extraction, RT-PCR analysis, and Southern hybridization with fluorescein-labeled internal probes followed by enhance chemiluminescence (ECL) detection. The fusion gene transcripts could be detected using 50 cells from FS materials in all cases and from 1 cell in 9 of 12 cases. For PS, a positive signal could be detected using 200 to 1000 cells in all cases, while weaker signals were detected using 50 cells in most cases. These results indicate that the fusion gene products from small round cell sarcomas can be detected by RT-PCR with 10 to 200 cells from FS and PS tissues. The sensitivity of RT-PCR with FS was 10- to 50-fold greater than with PS. These results also suggest that RT-PCR analysis for sarcoma fusion gene products can be successfully performed when only a few cells are available for analysis, although this is not recommended for routine clinical use.

Blotting, Southern↗
PubMed2004

Definition of a region of loss of heterozygosity at chromosome 11q23.3-25 in head and neck squamous cell carcinoma using laser capture microdissection technique.

To date, loss of heterozygosity (LOH) studies on HNSCC have had limited success in identifying a confined region of loss on chromosome 11q partially due to the heterogeneous nature of tumor tissue examined. Additionally, little is known about the role of the 11q allelic deletion in HNSCC tumorigenesis and current reports are conflicting. The aim of this study was to better define LOH at distal 11q by using combination of a pure cell population procured by laser capture microdissection (LCM) and subsequent sensitive PCR amplification of polymorphic microsatellites. This study analyzed HNSCC for LOH using a panel of 5 microsatellite markers spanning 11q23-25. Thirty-four paired DNA samples from tumor and autologous normal tissue were harvested by LCM technique to ensure a pure cell population for PCR amplification. Approximately 2000 to 3000 cells were procured from each sample. Twenty-one of 34 cases (62%, P < 0.001) showed LOH on at least one of the loci examined. The highest frequency of LOH was found at the 11q23.3-25 segment, with 44% at marker D11S968 and 35% at marker D11S1316. A distinct novel region of frequent LOH at 11q23.3-25, defined by D11S1316 and D11S968, was identified. No allelic loss was found in any normal squamous tissue samples. To study LOH in HNSCC, combination of pure cell population procurement by LCM and sensitive PCR provides a more accurate approach than the conventional method using a bulk of heterogeneous tissue. A novel region of LOH at 11q23.3-25 was defined. LOH in this region may harbor putative tumor suppressor gene(s) critical for HNSCC. Furthermore, these allelic losses were not found in any non-neoplastic squamous tissue samples, clarifying prior discrepant data.

Adult↗
PubMed2003

Differential protein analysis of spasomolytic polypeptide expressing metaplasia using laser capture microdissection and two-dimensional difference gel electrophoresis.

Full analysis of cellular protein constituents is a valuable tool in the evaluation of tissues. Traditional methods of evaluation, however, are time-consuming and difficult to reproduce. Two-dimensional difference gel electrophoresis (2D-DIGE), a recently developed proteomic system, affords the ability to compare and evaluate protein extracts from multiple sources. Coupled with laser capture microdissection (LCM), this technology is a powerful tool in comparing the protein profiles of separate pure cell populations. Proteins are labeled in vitro with reactive cyanine dyes that fluoresce at differential wavelengths, and after comigration on two-dimensional gels, differing protein populations become apparent. The unique aspect of this technology is the ability to identify and quantify proteins from separate preparations without issues of gel-to-gel differences. These techniques coupled with the systems for robotic acquisition of specific spots on the gel, tryptic digestion, and MALDI mass spectrometry permit identification of proteins differentially expressed in two pure cell populations. The authors used these new technologies to analyze the protein constituents of spasmolytic polypeptide expressing metaplasia (SPEM), a gastric mucosal metaplasia (fundic antralization or pseudopyloric metaplasia) that develops in the atrophic fundus mucosa of mice infected with Helicobacter felis and in humans infected with Helicobacter pylori. In addition, SPEM has been identified in the atrophic mucosa surrounding a high percentage of gastric adenocarcinomas and may represent a precursor lineage of malignancy. This technology recognized 28 differentially expressed proteins between SPEM and surface cells. Identification of novel SPEM-related proteins would allow the development of new immunohistochemical antibodies to further study this important metaplasia.

Animals↗
PubMedDate not supplied

Laser-assisted microdissection applied to frozen surgical pathologic specimens - methodological aspects on RT-PCR.

Laser-assisted microdissection has become a unique technique for an accurate gene-expression profiling analysis in human tissues. The introduction of this approach requires the development of a reliable, efficient, and reproducible procedure for tissue processing. We report a systematic evaluation of the different relevant steps required to obtain sufficient quantity and good quality RNA for reverse transcriptase-polymerase chain reaction when using frozen surgical pathologic tissues as starting material. We propose an optimized and very efficient method with respect to time and effort that can easily be put into practice in research laboratory as well as in any pathology laboratory.

Colon↗
PubMed2004

Comparison of ethanol versus formalin fixation on preservation of histology and RNA in laser capture microdissected brain tissues.

Although RNA can be retrieved from formalin-fixed, paraffin-embedded (FFPE) tissues, the yield is low, and the RNA is fragmented. Recent advances in gene expression profiling underscore the importance of identifying a fixative that preserves histology and mRNA. We demonstrated that, for immersion fixation of brains, 70% ethanol is superior to formalin for mRNA preservation. RNA yield from ethanol-fixed tissues was 70% of the yield from fresh frozen specimens, but only a negligible quantity was recovered from formalin-fixed tissues. RNA from ethanol-fixed brains showed integrity comparable to RNA from fresh frozen tissues, and RT-PCR using RNA from ethanol-fixed tissues was consistently successful. RNA from FFPE tissues composed of low-molecular weight fragments, and their use in RT-PCR failed repeatedly. The yield and quality of RNA from ethanol-fixed brains were unaffected after immersion at 4 degrees C for 2 weeks. In a blinded comparison to FFPE tissues, ethanol-fixed specimens were judged to show comparable histology and superior immunostaining. After laser capture microdissection (LCM), we failed to recover mRNA from FFPE tissues but retrieved mRNA from ethanol-fixed tissues for RT-PCR and cDNA microarray analysis. We conclude that 70% ethanol preserves RNA integrity and is suitable for expression profiling of brain tissues by LCM and cDNA microarray.

Animals↗