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At least 667 records · Page 37Linked to original sources
PubMed2005

Laser microdissection in clinical cardiovascular research.

Laser microdissection (LMD) is an accurate and fast method to procure pure populations of cells from complex, heterogeneous tissues under direct microscopic visualization. It can be applied to a wide range of cell preparation, including paraffin-embedded material. The morphology of the captured cells is retained, and DNA, RNA, and proteins can be extracted for molecular analysis. The potential applications of LMD to human cardiovascular research are multiple, including viral/autoimmune myocarditis and arteritis, atherosclerotic lesions, and myocardial and vascular cell proliferation and death. Molecular and genetic analysis of LMD-procured cells in cardiac and vascular tissues may provide a better understanding of several cardiac diseases.

Biopsy↗
PubMed2004

Partial sciatic nerve transection.

Partial sciatic nerve transection (PST) of the sciatic nerve of rats and mice is described as a model of painful neuropathy. The rationale for developing this model was to establish a simple partial nerve injury without application of foreign material. In contrast to the frequently used model of chronic constriction injury (CCI), PST allows to relate animal behavior and drug effects to endoneurial changes, undisturbed by major epineurial inflammation. PST is easy to perform in rats and mice and leads to reproducible pain-related behavior for 5 wk or longer.

Animals↗
PubMed2004

Laser-mediated microdissection as a tool for molecular analysis in arthritis.

Most current approaches to the analysis of gene expression in arthritic tissue samples are based on RNA isolated either from cultured synovial cells or from synovial biopsies. However, this strategy does not distinguish between specific gene expression profiles of cells originating from separate tissue areas. Therefore, we established the combination of laser-mediated microdissection and RNA arbitrarily primed polymerase chain reaction (RAP-PCR) for differential display to analyze profiles of gene expression in histologically defined areas of arthritic tissue. Cryosections derived from synovial tissue were used to obtain cell samples from different tissue areas using a microbeam laser microscope. RNA was isolated and analyzed using nested RAP-PCR to generate a fingerprint of the expressed gene sequences. Differentially expressed bands were isolated, cloned, and sequenced. Differential expression of identified sequences was confirmed by in situ hybridization and immunohistochemistry.

Arthritis↗
PubMed2005

Assessment of RET/PTC oncogene activation in thyroid nodules utilizing laser microdissection followed by nested RT-PCR.

Single palpable nodules of the thyroid gland are common in clinical practice; the majority of such lesions are benign. However, noninvasive thyroid nodules that exhibit borderline morphological signs of papillary cancer represent a diagnostic challenge. Rearrangements of the RET oncogene have been proposed as a marker for papillary thyroid cancer. In this chapter, methods for the analysis of the RET oncogene in laser microdissected papillary thyroid cancer tissue are described.

Biomarkers, Tumor↗
PubMed2005

Laser capture microdissection for analysis of macrophage gene expression from atherosclerotic lesions.

Macrophage foam cells are critical mediators in atherosclerosis plaque development. A better understanding of the in vivo transcript profile of foam cells during the formation and progression of lesions may lead to novel therapeutic interventions. Toward this goal, we demonstrate for the first time that foam cell-specific RNA can be purified from atherosclerotic arteries, a tissue of mixed cellular composition. Foam cells from apolipoprotein (apo) E-/- mice were isolated by laser capture microdissection (LCM); RNA was extracted and used for molecular analysis by real-time quantitative polymerase chain reaction. Compared to whole tissue, a significant enrichment of foam cell-specific RNA transcripts was achieved. Furthermore, to test the ability to quantify differences in gene expression in response to an inflammatory stimulus, apoE-/- mice were injected with lipopolysaccharide, after which the transcriptional induction of the inflammatory mediators, VCAM, ICAM, and MCP-1, was observed in lesional macrophage foam cell RNA. These approaches will facilitate the study of macrophage gene expression under various conditions of plaque formation, regression, and response to genetic and environmental perturbations.

Animals↗
PubMed2005

Analysis of pituitary cells by laser capture microdissection.

The anterior pituitary gland consists of a heterogeneous population of various cell types. To study a single cell type with a homogeneous cell population, one can perform laser capture microdissection (LCM). Because different pituitary cells have unique immunophenotypic profiles, it is possible to perform immunohistochemical staining before LCM (immuno-LCM) for the collection of a phenotypically homogeneous cell population. These techniques were developed and applied to dissociated anterior pituitary cells and cultured pituitary cells. When combined with reverse transcriptase polymerase chain reaction, it is possible to analyze gene expression in as few as one to 10 pituitary cells. We have used the immuno-LCM technique to prepare homogeneous populations of folliculostellate cells. These cells were analyzed for expression of peptides and receptors. Anterior pituitary hormones were not expressed by these cells. These results show the utility of immuno-LCM for cellular and molecular studies of gene expression.

Animals↗
PubMed2005

Use of laser capture microdissection together with in situ hybridization and real-time PCR to study distribution of latent herpes simplex virus genomes in mouse trigeminal ganglia.

The herpes simplex virus (HSV) frequently establishes a latent state in neurons, which can then be reactivated from the infected neurons. Quantifying the single-cell viral load is essential for understanding latency and reactivation of this virus. In this chapter the methods of laser capture microdissection and quantitative real-time polymerase chain reaction with in situ hybridization have been combined to determine the HSV copy number per neuron in latently infected trigeminal ganglia. The distribution of latent herpes simplex genomes at the individual cell level has been deteremined and the relationship of the number of latent genomes to the expression of latency-associated transcripts established.

Animals↗
PubMed2005

Laser capture microdissection and PCR for analysis of human papilloma virus infection.

Human papilloma virus (HPV) infection is considered one of the main factors involved in the pathogenesis of endocervical adenocarcinoma. However, the cellular location of HPV in this type of tumor is controversial. We have developed a method to determine the presence of HPV type 16 in endocervical cancer cells using laser capture microdissection followed by DNA extraction and qualitative polymerase chain reaction. Our results show that HPV type 16 is present in endocervical adenocarcinoma cells.

Adenocarcinoma↗
PubMed2006

Proteomics and laser microdissection.

Two-dimensional gel electrophoresis (2-DE) combined with protein identification by mass spectrometry (MS) is currently the method of choice in the majority of proteomic projects. Novel gel-free technologies have been developed but 2-DE remains the technique of choice for quantitative expression profiling of large sets of complex protein mixtures such as whole cell/tissue lysates. Solubilized proteins are separated in the first dimension according to their charge properties (isoelectric point, pI) by isoelectric focusing (IEF) under denaturing conditions, followed by their separation in the second dimension by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), according to their relative molecular mass (Mr). 2-DE can resolve more than 5000 proteins simultaneously (approximately 2000 proteins routinely) and can detect less than 1 ng of protein per spot. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or posttranslational modifications. In this chapter we describe the various steps in the 2-DE proteomics workflow, namely sample preparation, solubilization, 2-D gel electrophoresis, protein detection and visualization, and protein identification by mass spectrometry. The use of 2-DE in conjunction with laser microdissection microscopy is presented and discussed.

Animals↗
PubMed2005

In situ isolation of immunoglobulin sequences expressed by single tumor-infiltrating B cells using laser-assisted microdissection.

The isolation of fully human monoclonal antibodies (MAb) against tumor targets has to date relied largely on combinatorial library-based antibody display techniques, which generally require lengthy antigen selection procedures due to a low frequency of clones expressing compatible heavy (VH) and light chain (VL) variable genes. Here we describe a method to directly isolate immunoglobulin sequences in situ from antibody-producing cells infiltrating human tumor tissue. Single B cells and plasma cells infiltrating cervical cancer were microdissected from tissue sections using laser-assisted microscopy, and VH and VL expressed by each individual cell amplified using nested reverse transcriptase- polymerase chain reaction (RT-PCR), thus retaining the native VH and VL pairing. Sequencing analysis determined that the isolated cells expressed functional immunoglobulin variable genes, consistent with an antitumor antibody response. The immunoglobulin sequences can be reassembled as Fab or scFv fragments using conventional recombinant antibody expression plasmids. This method will allow a more direct assessment of the humoral immune response to cancer, and the potential identification of novel human therapeutic cancer antibodies.

Antibodies, Monoclonal↗
PubMed2004

Clinico-pathological significance of cell-type-specific loss of heterozygosity on chromosome 7q21: analysis of 318 microdissected thyroid lesions.

A careful pathological examination often reveals the presence of different lesions at various stages of tumor progression and invasion, even in those thyroid glands presenting with solitary nodules. Each thyroid lesion is composed of many different cell types, reflecting the marked heterogeneity of normal thyroid tissue. Among the different chromosome regions altered in thyroid tumors, 7q21 appears to be specifically involved in malignant tumors, especially of the follicular type. This study was conducted to analyze the loss of heterozygosity (LOH) pattern at 7q21 in pure populations of cells from each single lesion harbored in surgically removed thyroid glands, and to evaluate its clinical significance. One hundred and forty-two thyroid glands were examined, all showing, as a common trait, a goitrous appearance associated with one single lesion in 114 cases and with more than one in the remaining 28 cases. A total number of 318 lesions was analyzed, consisting of 142 goiters (TG), 48 hyperplasias (TH), 80 adenomas (TA) and 48 carcinomas (TC). Five different types of cells were isolated by laser capture microdissection from each lesion. DNA was analyzed by PCR and polyacrylamide gel electrophoresis in search of LOH affecting five microsatellite markers, D7S660, D7S630, D7S492, D7S657, and D7S689. We detected LOH at 7q21 not only in thyroid malignant tumors but also in benign lesions. Allelic loss occurred exclusively in dark nucleus and eosinophilic cytoplasm cells, commonly observed in the follicular type of lesions. In these types of lesions allelic loss frequency increases along with neoplastic transformation (9% in TG, 41% in TH, 68% in TA and 100% in TC), and is directly correlated with thyroid gland volume as well as with the presence of multiple lesions. The highest LOH rate was observed for D7S492, indicating that the recurrent region of deletion was localized at the corresponding genetic locus at 7q21.2, in the same position where the common fragile site FRA7E was previously mapped. LOH at this locus represents an early event in the development of follicular TC and is associated with intense growth of thyroid glands.

Adenocarcinoma, Follicular↗
PubMed2005

Osteoclast-like cells express receptor activity modifying protein 2: application of laser capture microdissection.

Receptor activity modifying proteins (RAMPs) act as receptor modulators that determine the ligand specificity of receptors for the calcitonin (CT) family. The purpose of this study was to analyze the expression of RAMPs in osteoclast-like cells using the laser capture microdissection (LCM) technique. Mouse bone marrow and spleen cells were co-cultured on a film designed for LCM. After 10 days, 250 osteoclast-like cells were captured using the LCM system. Total RNA from these cells was used to synthesize cDNA and RT-PCR analysis was performed. Osteoclast-like cells expressed CT receptor (CTR), CT receptor-like receptor (CRLR) and RAMP2, but did not express RAMP1 or RAMP3. These results indicated (1) that a pure population of osteoclast-like cells can be prepared by LCM and gene expression of this population can be analyzed by RT-PCR and (2) that RT-PCR shows that osteoclast-like cells express RAMP2, CTR and CRLR, suggesting the potential for adrenomedullin binding to osteoclast-like cells. This is the first report that osteoclast-like cells express RAMP2.

Animals↗
PubMed2004

Analysis of estrogen-regulated genes in mouse uterus using cDNA microarray and laser capture microdissection.

The steroid hormone, estrogen, plays an important role in various physiological events which are mediated via its nuclear estrogen receptors, ERalpha and ERbeta. However, the molecular mechanisms that are regulated by estrogen in the uterus remain largely unknown. To identify genes that are regulated by estrogen, the ovariectomized mouse uterus was exposed to 17beta-estradiol (E2) for 6 h and 12 h, and the data were analyzed by cDNA microarray. The present study confirms previous findings and identifies several genes with expressions not previously known to be influenced by estrogen. These genes include small proline-rich protein 2A, receptor-activity-modifying protein 3, inhibitor of DNA binding-1, eukaryotic translation initiation factor 2, cystatin B, decorin, secreted frizzled-related protein 2, integral membrane protein 2B and chemokine ligand 12. The expression patterns of several selected genes identified by the microarray analysis were confirmed by RT-PCR. In addition, laser capture microdissection (LCM) was conducted to determine the expression of selected genes in specific uterine cell types. Analysis of early and late responsive genes using LCM and cDNA microarray not only suggests direct and indirect effects of E2 on uterine physiological events, but also demonstrates differential regulation of E2 in specific uterine cell types. These results provide a basic background on global gene alterations or genetic pathways in the uterus during the estrous cycle and the implantation period.

Animals↗
PubMed2005

Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization.

Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells. By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S rRNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria within the tissue with molecular analysis of 16S rRNA gene or other genes of interest. This method is widely applicable for the identification of noncultivable bacteria and their gene pool from formalin-fixed paraffin-embedded tissue samples.

Brachyspira↗
PubMed2006

Validation of a novel ultra-short immunolabeling method for high-quality mRNA preservation in laser microdissection and real-time reverse transcriptase-polymerase chain reaction.

Laser microdissection allows isolation of tiny samples from tissue sections for analysis of gene expression by real-time quantitative polymerase chain reaction (PCR). Although immunohistochemical labeling is often required to identify target structures, it drastically degrades mRNA so that shortened protocols are needed. Here, we present a novel method that allows fluorescence double labeling to be performed in only one incubation of 5 minutes. Fab fragments directly coupled to fluorochromes are linked to primary antibodies before these complexes are applied to sections. We quantified the influences of fixatives, labeling solutions, and incubation time on the mRNA yield and compared our method with previously proposed protocols. While tissue components, ie, vimentin and Ki67 antigen, were sufficiently stained after only 5 minutes of incubation, the new method produced a minute loss of mRNA that did not significantly differ from that of untreated sections. In contrast, incubation times of 15 and 30 minutes reduced the mRNA yield by 99.8 to 99.9%. Furthermore, incubation periods longer than 5 minutes critically affected the ratio between the target and housekeeping genes tested by factors of up to 10.6. In conclusion, the novel method described here reduces mRNA loss and potential ratio shifts to a level that does not significantly differ from that of unlabeled samples.

Animals↗
PubMed2003

Gene expression in the rhesus monkey endometrium: differential display and laser capture microdissection.

The primate endometrium is a complex heterogeneous tissue that requires proper maturation to achieve a hospitable environment for implantation. Endometrial differentiation and maturation is primarily controlled through the action of progesterone during the secretory phase. Many of the genes and gene networks that are involved in this process are likely to be induced or inhibited in a temporal, spatial, and cell-type specific context within the endometrium. We have used several approaches to address these latter issues in the rhesus monkey endometrium. The use of differential display with hormonally distinct endometrial cDNA populations prepared from artificially controlled menstrual cycles has allowed us to identify different P-dependent mRNA regulatory patterns during simulated secretory phases. In addition, we have coupled differential display with laser capture microdissection to further study region and cell-type specific changes in the primate endometrium.

Animals↗
PubMed2004

Expression of survivin in primary and metastatic gastric cancer cells obtained by laser capture microdissection.

AIM: Survivin, a recently identified member of the inhibitor of apoptosis protein family, is expressed during development and in various human cancers. However, its expression in normal tissues and clinical relevance in cancers are still debated. In the present study, we analyzed the expression of the survivin gene in human primary and metastatic gastric cancer cells as well as in paired epithelial cells from normal gastric mucosa by means of a novel laser capture microdissection (LCM) technique coupled with reverse transcription-polymerase chain reaction (RT-PCR). METHODS: Thirty patients who had undergone gastrectomy with lymph node dissection for gastric cancer without preoperative treatments were included. Neoplastic tissue, metastatic lymph nodes, and apparently uninvolved normal tissue were collected from each patient. LCM-captured "pure" cell groups were respectively subjected to RT-PCR analysis with primers specific for the survivin gene. RESULTS: Of the paired samples from 30 gastric cancer patients studied, 24 (80%) primary gastric cancer cell groups and 7 (23%) adjacent morphologically "normal" gastric epithelial cell groups were shown to have a detectable survivin expression. There was a statistically significant difference in suvivin expression between these two groups (P<0.01). Meanwhile, 95% (19/20) of the metastatic gastric cancer cell groups from lymph nodes had a clear expression of te survivin gene. However, no significant correlation between survivin expression and clinicopathological features of gastric cancer was observed in the present study. CONCLUSION: Survivin expression is present in the majority of gastric cancer cell groups obtained by LCM techniques. The high expression rate in metastatic lesions suggests a possible role of survivin in cancer invasiveness and metastasis. It may contribute to the detection of gastric cancer micrometastasis as a potential molecular marker. In addition, the high expression percentage renders survivin a potential target in the therapy for gastric cancer.

Adult↗
PubMed2005

Gene expression profiling of gastric cancer by microarray combined with laser capture microdissection.

AIM: To examine the gene expression profile of gastric cancer (GC) by combination of laser capture microdissection (LCM) and microarray and to correlate the profiling with histological subtypes. METHODS: Using LCM, pure cancer cells were procured from 45 cancerous tissues. After procurement of about 5000 cells, total RNA was extracted and the quality of RNA was determined before further amplification and hybridization. One microgram of amplified RNA was converted to cDNA and hybridized to cDNA microarray. RESULTS: Among 45 cases, only 21 were qualified for their RNAs. A total of 62 arrays were performed. These included 42 arrays for cancer (21 cases with dye-swab duplication) and 20 arrays for non-tumorous cells (10 cases with dye-swab duplication) with universal reference. Analyzed data showed 504 genes were differentially expressed and could distinguish cancerous and non-cancerous groups with more than 99% accuracy. Of the 504 genes, trefoil factors 1, 2, and 3 were in the list and their expression patterns were consistent with previous reports. Immunohistochemical staining of trefoil factor 1 was also consistent with the array data. Analyses of the tumor group with these 504 genes showed that there were 3 subgroups of GC that did not correspond to any current classification system, including Lauren's classification. CONCLUSION: By using LCM, linear amplification of RNA, and cDNA microarray, we have identified a panel of genes that have the power to discriminate between GC and non-cancer groups. The new molecular classification and the identified novel genes in gastric carcinogenesis deserve further investigations to elucidate their clinicopathological significance.

Gene Expression Profiling↗