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At least 559 records · Page 31Linked to original sources
PubMed2005

Karyotypic evolution of a novel cervid satellite DNA family isolated by microdissection from the Indian muntjac Y-chromosome.

A minilibrary was constructed from DOP-PCR products using microdissected Y-chromosomes of Indian muntjac as DNA templates. Two microclones designated as IM-Y4-52 and IM-Y5-7 were obtained from negative screening of all three cervid satellite DNAs (satellites I, II, and IV). These two microclones were 295 and 382 bp in size, respectively, and shared approximately 70% sequence homology. Southern blot analysis showed that the IM-Y4-52 clone was repetitive in nature with an approximately 0.32-kb register in HaeIII digest. Sequence comparison revealed no similarities to DNA sequences deposited in the GenBank database, suggesting that the microclone sequences were from a novel satellite DNA family designated as cervid satellite V. A subclone of an Indian muntjac BAC clone which screened positive for IM-Y4-52 had a 3,325-bp insert containing six intact monomers, four deleted monomers, and two partial monomers. The consensus sequence of the monomer was 328 bp in length and shared more than 80% sequence homology with every intact monomer. A zoo blot study using IM-Y4-52 as a probe showed that the strong hybridization with EcoRI digested male genomic DNA of Indian muntjac, Formosan muntjac, Chinese muntjac, sambar deer, and Chinese water deer. Female genomic DNA of Indian muntjac, Chinese water deer, and Formosan muntjac also showed positive hybridization patterns. Satellite V was found to specifically localize to the Y heterochromatin region of the muntjacs, sambar deer, and Chinese water deer and to chromosome 3 of Indian muntjac and the X-chromosome of Chinese water deer.

Animals↗
PubMed2006

Probing the W chromosome of the codling moth, Cydia pomonella, with sequences from microdissected sex chromatin.

The W chromosome of the codling moth, Cydia pomonella, like that of most Lepidoptera species, is heterochromatic and forms a female-specific sex chromatin body in somatic cells. We collected chromatin samples by laser microdissection from euchromatin and W-chromatin bodies. DNA from the samples was amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and used to prepare painting probes and start an analysis of the W-chromosome sequence composition. With fluorescence in situ hybridization (FISH), the euchromatin probe labelled all chromosomes, whereas the W-chromatin DNA proved to be a highly specific W-chromosome painting probe. For sequence analysis, DOP-PCR-generated DNA fragments were cloned, sequenced, and tested by Southern hybridization. We recovered single-copy and low-copy W-specific sequences, a sequence that was located only in the W and the Z chromosome, multi-copy sequences that were enriched in the W chromosome but occurred also elsewhere, and ubiquitous multi-copy sequences. Three of the multi-copy sequences were recognized as derived from hitherto unknown retrotransposons. The results show that our approach is feasible and that the W-chromosome composition of C. pomonella is not principally different from that of Bombyx mori or from that of Y chromosomes of several species with an XY sex-determining mechanism. The W chromosome has attracted repetitive sequences during evolution but also contains unique sequences.

Animals↗
PubMed2006

Sex-specific fluorescent labelling of cells for laser microdissection and DNA profiling.

Sex-specific isolation of cells from mixtures would greatly facilitate forensic casework. Thus, male and female cell mixtures were marked with a fluorescent X/Y-probe CEP X SpectrumOrange/Y SpectrumGreen DNA probe kit for fluorescence in situ hybridization, and single cells were isolated via laser microdissection (LMD). DNA profiling of LMD isolated, hybridized cells showed usable short tandem repeat profiles for at least 20 cells, which are comparable with results from other studies. To simulate casework samples, the method was also optimized for air-dried samples.

Blood Cells↗
PubMed2005

Digoxigenin labelling and laser capture microdissection of male cells.

Laser capture microdissection (LMD) is a relatively new technique for the isolation of single cells. The application in forensic investigations has become more and more widespread, especially to select spermatozoa out of mixtures with vaginal cells. In particular in cases with low numbers of sperm it could be profitable to isolate all male cells (e.g. sperm and male epithelial cells) instead of focussing on the sperm only. Therefore, the specific labelling and detection of the male cells in a male/female cell mixture is necessary. In order to label all cells carrying a Y-chromosome we used a digoxigenin labelled chromosome Y hybridisation probe (Q Biogen). The stained cells were isolated with the SL microCut LMD system from Molecular Machines & Industries AG (MMI). At least ten diploid male cells were required to obtain a partial STR profile, with 20 cells, a full profile could be obtained.

Chromosomes, Human, Y↗
PubMed2005

An in vitro model for studying vascular injury after laser microdissection.

We have developed an in vitro model for studying vascular injury. After 7-10 days in a three-dimensional collagen gel culture, capillary-like tubes were formed in the collagen gels. We injured these capillary-like tubes with a laser microdissection system or a scrape method with razors and then examined the collagen gel culture by phase contrast and electron microscopy. After laser injury, profuse necrotic cells were observed around the injured capillary-like tubes and within the tubular lumen compared to the razor injury. We then isolated total RNA from these cultures and prepared cDNA for investigations by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Quantitative real time RT-PCR revealed the up-regulation of transcription factor early growth response-1 (Egr-1) after both laser and razor injury, accompanied by the up-regulation of fibroblast growth factor-2 (FGF-2), a proangiogenic factor downstream of Egr-1. The effective laser energy is concentrated on the minute focal spot only. These methods provide a useful in vitro model for studying vascular injury.

Animals↗
PubMed2006

New approach to in situ quantification of ovarian gene expression in rat using a laser microdissection technique: relationship between follicle types and regulation of inhibin-alpha and cytochrome P450aromatase genes in the rat ovary.

The recently developed laser microdissection (LMD) technique makes it possible to quantify local gene expression in the target cells of various tissues. Using the LMD technique, this study aimed at comparing the amounts of mRNAs encoding the inhibin-alpha subunit and cytochrome P450 aromatase (P450(arom)) in granulosa cells between preantral and antral follicles in immature rat ovaries. Serial frozen sections of the ovaries from 24-day-old female Wistar rats were made and 30 healthy preantral (100-200 microm maximum diameter) and ten healthy antral ( > 300 microm maximum diameter) follicles were selected in each ovary based on morphological examinations, including immunohistochemistry for inhibin-alpha, in sections adjacent to those used for LMD. The amounts of mRNAs encoding inhibin-alpha subunit and P450(arom) were quantified by real-time polymerase chain reaction (PCR). While the amount of P450(arom) mRNA in the granulosa cell layers from the antral follicles was about 12-times higher than that in the preantral follicles, no difference in the amount of inhibin-alpha mRNA was found between these two types of follicles. Thus, the LMD technique allowed the in situ quantification of gene expression in the ovary and revealed that each granulosa cell expresses a stable amount of inhibin-alpha subunit mRNA independently of antral formation in immature rat ovaries.

Animals↗
PubMed2006

Laser microdissection combined with immunohistochemistry on serial thin tissue sections: a method allowing efficient mRNA analysis.

Laser microdissection (LMD) with subsequent reverse transcription-PCR analysis is a powerful histochemical technique subserving the molecular characterization of specific cell types. We developed an efficient method for selective sampling of specific cell populations using immunohistochemistry coupled with LMD. The cerebral cortex of adult rats was cut into serial thin sections. Some sections were immunostained for parvalbumin. The adjacent sections were mounted on Cell Support Film for LMD and stained with neutral red. By comparison of the two adjacent sections, neuronal profiles representing parts of parvalbumin-immunopositive somata were identified in the neutral red-stained sections. These neuronal profiles were safely captured with LMD and analyzed on reverse transcription-PCR using extracted RNA. The method presented here can be applied to cell-type-specific characterizations using fixed cells under RNase-free conditions.

Animals↗
PubMed2006

Preparative laser capture microdissection and single-pot cell wall material preparation: a novel method for tissue-specific analysis.

In adaptation to their function the walls of plant cell display tissue-specific variations of composition according to their developmental stage, cell type and stress of various origin. It is therefore important to obtain a precise analytical data describing the cell wall composition with respect to these different factors. In the present work, laser capture microdissection (LCM) was used for isolating different tissues from the stem of Urtica dioica L. at a semi-preparative scale. The technique was associated for the first time to a one-pot sequential cell wall preparation and hydrolysis for the carbohydrate analysis of each cell type. The results demonstrate that the combination of LCM and micro-analytical methods can provide individual cell type composition and should improve our knowledge of the biochemical diversity of cell walls in plants. This approach will be of potential interest for the understanding of the effects of stress or genetic engineering on the composition of the cell walls.

Carbohydrates↗
PubMed2003

Gene expression profiles of microdissected pancreatic ductal adenocarcinoma.

In a search for new molecular markers of pancreatic ductal adenocarcinoma (PDAC), we compared the gene expression profiles of seven pancreatic carcinomas and one carcinoma of the papilla Vateri with those of duct cells from three non-neoplastic pancreatic tissues. In addition, the human pancreatic duct cell line and five PDAC cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2, HPAF) were examined. RNA was extracted from microdissected tissue or cultured cell lines and analysed using a custom-made Affymetrix Chip containing 3023 genes, of which 1000 were known to be tumour associated. Hierarchical clustering revealed 81 differentially expressed genes. Of all the genes, 26 were downregulated in PDAC and 14 were upregulated in PDAC. In PDAC cell lines versus normal pancreatic duct cells, 21 genes were downregulated and 20 were upregulated. Of these 81 differentially expressed genes, 15 represented human genes previously implicated in the tumourigenesis of PDAC. From the genes that were so far not known to be associated with PDAC tumorigenesis, we selected ADAM9 for further validation because of its distinct overexpression in tumour tissue. Using immunohistochemistry, the over-expressed gene, ADAM9, was present in 70% of the PDACs analysed. In conclusion, using microarray technology we were able to identify a set of genes whose aberrant expression was associated with PDAC and may be used to target the disease.

Adenocarcinoma↗
PubMed2005

Loss of p16INK4A expression is associated with allelic imbalance/loss of heterozygosity of chromosome 9p21 in microdissected synovial sarcomas.

Deletions of the short arm of chromosome 9 have been observed in many tumours and cell lines. This chromosomal region is frequently targeted during malignant transformation because it contains at least two known tumour suppressor genes: p16(INK4) and p15(INK4B). p16(INK4A) acts as a negative cell cycle regulator by inhibiting G1 cyclin-dependent kinases that phosphorylate the retinoblastoma protein and therefore block the progression of the cell cycle from G1 to S phase. The role of p16(INK4A) in the development of synovial sarcoma has not been comprehensively investigated. Ten samples of synovial sarcomas were examined for allelic imbalance/loss of heterozygosity (AI/LOH) of the 9p region and p16 protein expression. DNA was isolated from microdissected sections of normal and tumour cells, amplified by polymerase chain reaction and analysed for AI/LOH by using six microsatellite markers that map to the 9p region. Immunohistochemistry for p16 expression was done. AI/LOH with at least one microsatellite marker on 9p21 was detected in six of ten samples. The most frequent allelic deletions were observed within the coding sequence of p16(INK4A). Loss of p16 immunoreactivity was detected in eight samples, six of which showed evidence of alterations at 9p21 region. These findings suggest a possible role of loss of p16(INK4A) in the development of synovial sarcoma.

Chromosomes, Human, Pair 9↗
PubMed2006

Analysis of gene expression identifies candidate molecular markers in nasopharyngeal carcinoma using microdissection and cDNA microarray.

PURPOSE: Microarray analysis was used to bring a comprehensive insight into underlying molecular mechanisms and obtain a whole assessment of aberrant gene expression in nasopharyngeal carcinoma (NPC). METHODS: Combined with microdissection, gene expression profiles in 23 NPCs and 10 nontumor nasopharyngeal epithelial tissue samples were analyzed. RESULTS: Gene expression patterns suggested the dysregulation of the GTP/GDP-bound Ras cycle and an abnormal hyperactivity of cell cycle in NPC. Alterations in the WNT pathway suggest that this pathway may be activated in NPC. A 6-feature weighted-voting model was chosen because it represented the main characteristics of NPCs and predicted NPCs most accurately from the nontumor tissues (33 of 34 correct calls; 97.1% accuracy, Fisher's exact test, P value = 8.389 x 10(-8)). CONCLUSIONS: The data generated in this study represent a comprehensive list of genes aberrantly regulated in NPC. The 6-feature weighted-voting model may provide an extensive list of potential molecular markers for early diagnosis.

Biomarkers, Tumor↗
PubMed2004

Needle in a haystack: microdissecting the proteome of a tissue.

Laser-assisted microdissection is a recent technology that enables cells to be harvested from tissue sections. Proteins can be extracted from the dissected cells for molecular analysis. This enables the analysis of proteins in specific cell types in an in vivo system. Although quantities of protein obtained from the dissected material can be small, it is possible to use established methods such as Western Blotting and 2D-PAGE, as well as newer technologies such as SELDI-MS, to analyse the proteins. This review describes the applications and technical considerations for using laser-assisted dissected cells in proteomics research.

Animals↗
PubMed2005

Downregulation of apoptosis revealed by laser microdissection and cDNA microarray analysis of related genes in rat liver preneoplastic lesions.

Regulation of cell proliferation and apoptosis plays a key role in tumor development. To elucidate mechanisms underlying hepatocarcinogenesis, patterns of gene expression, including apoptosis-related genes, were compared between glutathione S-transferase placental form (GST-P)-positive preneoplastic foci and surrounding tissue in the rat. Lesions were induced with a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg bw) and then 100 ppm 2-acetylaminofluorene (2-AAF)-containing diet combined with two-thirds partial hepatectomy. Frozen sections of the livers were applied for immunohistochemical staining of GST-P, and both positive foci and surrounding negative areas were harvested by laser microdissection. Total RNAs were extracted and amplified with T7 polymerase to allow gene expression analysis by cDNA microarray assays. In the GST-P-positive foci, altered levels were observed for many genes, mostly related to metabolism or catalysis, with increased expression of testosterone-repressive prostate message-2 (TRPM-2), which is reported to act as a protective factor against apoptosis, and decreased expression of thymus-expressed acidic protein (TEAP), which is considered to promote apoptosis. The results indicate that rat liver preneoplastic lesions might be protected against apoptosis and that the approach adopted is useful for clarification of mechanisms underlying hepatocarcinogenesis.

Alkylating Agents↗
PubMed2005

cDNA microarray analysis after laser microdissection in proliferating islets of partially pancreatectomized mice.

With islet transplantation having grown in popularity since the introduction of the Edmonton protocol, how to secure an unlimited source of islets has become an urgent problem. To resolve this problem, techniques to induce or proliferate islets are urgently required. To achieve this goal, gene expression analysis using a cDNA microarray in islets of partially pancreatectomized mice, in which the remaining islets regenerate and proliferate with insulin secretion and glucose responsiveness, provides us with valuable information. However, those experiments have two critical problems: first, how to selectively collect the regenerating or proliferating islets, and second, the shortage of total RNA extracted from one islet for a microarray analysis. A useful system was thus designed which combined laser microdissection, cDNA amplification by SMART PCR, which can maintain the relative expression profile of transcripts throughout reactions, and a cDNA microarray. Furthermore, this system is expected to contribute to future studies regarding not only islet regeneration but also the function of the islet itself, and this system may also be applicable to many other types of endocrine tissue. In this review, the details of this system are presented and discussed.

Animals↗
PubMed2006

Expression of decorin and collagens I and III in different layers of human skin in vivo: a laser capture microdissection study.

Extracellular matrix (ECM) organization is a complex process that requires the coordinated efforts of many molecules. For the regulation of collagen fiber diameter, the proteoglycan decorin appears to be of major relevance. To investigate the role of decorin in the process of (photo-)aging in more detail, full-thickness punch biopsies were isolated from human buttock skin. Single exposure with two minimal erythemal doses of solar simulated irradiation caused down-regulation of decorin mRNA in young (n = 5) and old subjects (n = 5) after 24 h. Interestingly, decorin mRNA was elevated with age. To test the hypothesis that a decreased collagen-to-decorin-ratio impairs collagen structure we also investigated collagens I and III gene expression. Both were down-regulated with increasing age and after single UV-irradiation. As determined by laser capture microdissection-quantitative real time-Polymerase chain reaction (n = 11), decorin is mostly present in the reticular dermis while being absent from the papillary dermis. Minor expression was also observed in the epidermis. However, in contrast to full-thickness skin biopsies age-dependent changes in collagens I, III, and decorin expression could not be observed with this methodology indicating technical limitations. Together with our finding that collagens I and III mRNA are similarly expressed in the reticular and papillary dermis and are down-regulated by UV, our studies support the idea of a major role of decorin in ECM organization. Altered expression of decorin mRNA in the different dermal strata and a decrease in the collagen-to-decorin ratio inflicted by both age and ultraviolet irradiation possibly affect collagen bundle diameter and subsequently the mechanical properties of human skin.

Adult↗
PubMed2005

Laser capture microdissection (LCM) and expression analyses of Glycine max (soybean) syncytium containing root regions formed by the plant pathogen Heterodera glycines (soybean cyst nematode).

Roots of soybean, Glycine max cv. Kent L. Merr., plants susceptible to the soybean cyst nematode (SCN), Heterodera glycines Ichinohe, were inoculated and allowed to develop feeding sites (syncytia) for 8 days. Root samples enriched in syncytial cells were collected using laser capture microdissection (LCM). RNA was extracted and used to make a cDNA library and expressed sequence tags (ESTs) were produced and used for a Gene Ontology (GO) analysis. RT-PCR results indicated enhanced expression of an aquaporin (GmPIP2,2), alpha-tubulin (GmTubA1), beta-tubulin (GmTubB4) and several other genes in syncytium-enriched samples as compared to samples extracted from whole roots. While RT-PCR data showed increased transcript levels of GmPIP2,2 from LCM tissue enriched in syncytial cells, in situ hybridization showed prominent GmPIP2,2 hybridization to RNA in the parenchymal cells tightly juxtaposed to the syncytium. Immunolocalization indicated stronger alpha-tubulin signal within the syncytium as compared to surrounding tissue. However, alpha-tubulin labeling appeared diffuse or clumped. Thus, LCM allowed for the isolation of tissue enriched for syncytial cells, providing material suitable for a variety of molecular analyses.

Amino Acid Sequence↗
PubMed2004

Detection of K-ras and p53 mutations in sputum samples of lung cancer patients using laser capture microdissection microscope and mutation analysis.

Mutations in the p53 tumor suppressor gene and the K-ras oncogene have been frequently found in sputum and bronchoalveolar lavage (BAL) samples of lung cancer patients and other patients prior to presenting clinical symptoms of lung cancer, suggesting that they may provide useful biomarkers for early lung cancer diagnosis. However, the detection of these gene mutations in sputum and BAL samples has been complicated by the fact that they often occur in only a small fraction of epithelial cells among sputum cells and, in the case of p53 gene, at many codons. In this study, sputum cells were collected on a filter membrane by sputum cytocentrifugation and morphologically analyzed. Epithelial cells were selectively taken by using a laser capture microdissection microscope and analyzed by polymerase chain reaction (PCR) and single-stranded conformational polymorphism (SSCP) for p53 mutations and by PCR and denaturing gradient gel electrophoresis (DGGE) for K-ras mutations. This method was used to analyze sputum of 15 Chinese women with lung cancer from Xuan Wei County, China and detected mutations in sputum of 7 (46.7%) patients, including 5 patients with p53 mutations, 1 patient with a K-ras mutation, and 1 patient with K-ras and p53 mutations. For comparison, only two of the mutations were detected by conventional methods. Therefore, the laser capture/mutation analysis method is sensitive and facilitates the detection of low-fraction mutations occurring throughout the p53 and K-ras genes in sputum of lung cancer patients. This method may be applicable to the analysis of epithelial cells from clinically normal sputum or BAL samples from individuals with a high risk for developing lung cancer.

Biomarkers, Tumor↗
PubMed2005

Analysis of connective tissues by laser capture microdissection and reverse transcriptase-polymerase chain reaction.

Studies of gene expression from bone, cartilage, and other tissues are complicated by the fact that their RNA, collected and pooled for analysis, often represents a wide variety of composite cells distinct in individual phenotype, age, and state of maturation. Laser capture microdissection (LCM) is a technique that allows specific cells to be isolated according to their phenotype, condition, or other marker from within such heterogeneity. As a result, this approach can yield RNA that is particular to a subset of cells comprising the total cell population of the tissue. This study reports the application of LCM to the gene expression analysis of the cartilaginous epiphyseal growth plate of normal newborn mice. The methodology utilized for this purpose has been coupled with real-time quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) to quantitate the expression of certain genes involved in growth plate development and calcification. In this paper, the approaches used for isolating and purifying RNA from phenotypically specific chondrocyte populations of the murine growth plate are detailed and illustrate and compare both qualitative and quantitative RT-PCR results. The technique will hopefully serve as a guide for the further analysis of this and other connective tissues by LCM and RT-PCR.

Animals↗