Laser Capture Microdissection Research Papers (original) (raw)

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- Analytical Chemistry, Lasers, Laser Capture Microdissection, Microdissection

Cytomics aims to determine the molecular phenotype of single cells. Within the context of the -omics, cytomics allows the investigation of multiple biochemical features of the heterogeneous cellular systems known as the cytomes. Cytomics can be considered as the science of single cell-based analyses that links genomics and proteomics with the dynamics of cell and tissue function, as modulated by external inXuences. Inherent to cytomics are the use of sensitive, scarcely invasive, Xuorescence-based multiparametric methods and the event-integrating concept of individual cells to understand the complexity and behaviour of tissues and organisms. Among cytomic technologies, Xow cytometry, confocal laser scanning microscopy and laser capture microdissection are of great relevance. Other recent technologies based on single cell bioimaging and bioinformatic tools become important in drug discovery and toxicity testing, because of both highcontent and high-troughput. The multiparametric capacity of cytomics is very useful for the identiWcation, characterization and isolation of stem cell populations. In our experience, Xow cytometry is a powerful and versatile tool that allows quantitative analysis of single molecules, prokaryotic and eukaryotic cells for basic, biotechnological, environmental and clinical studies. The dynamic nature of cytomic assays leads to a real-time kinetic approach based on sequential examination of diVerent single cells from a population undergoing a dynamic process, the in Xuxo level. Finally, cytomic technologies may provide in vitro methods alternative to laboratory animals for toxicity assessment.

- by Laura Diaz
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- Pharmacology, Toxicology, Kinetics, Flow Cytometry

Pyramidal neurons in hippocampal subregions are selectively vulnerable in certain disease states. To investigate, we tested the hypothesis that selective vulnerability in human hippocampus is related to regional differences in neuronal cell death and cell receptor gene expression in CA1 vs. CA3 subregions. We used laser capture microdissection to remove approximately 600 CA1 and 600 CA3 pyramidal neurons each from five fresh-frozen normal post-mortem brains, extracted total RNA and double-amplified mRNA. This was reverse transcribed and labeled for hybridization onto human cDNA array chips containing probes to 10,174 genes and unknown ESTs. RNA from additional microdissections was pooled for replicate hybridizations and quantitative RT-PCR validation. Gene expression differences were few ( < 1%). We found 43 enriched genes in CA1 neuronal samples that included peripheral benzodiazipine receptor-associated protein, nicotinic cholinergic receptor, two chemokine receptors (CCR1 and CCR5) and several transcriptional factors. We found 17 enriched genes in the CA3 neuronal samples that included fibroblast growth factor receptor and prostaglandin-endoperoxide synthase 1. We found no differential gene expression for 23 calcium channel proteins; nine transporter proteins; 55 cell death and apoptotic regulator proteins; and an additional 497 cell receptors, including 24 glutamate receptors. Quantitative RT-PCR of four differentially expressed genes confirmed the microarray data. The results confirm the ability to examine gene expression profiles in microdissected neurons from human autopsy brain. They show only minor gene expression differences between two distinct neuronal populations in the hippocampus and suggest that selective hippocampal vulnerability is due to factors other than intrinsic differential expression in glutamate receptors and cell death genes. D

- by Richard Bookman
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- Cognitive Science, Gene expression, Neuronal cell death, Hippocampus

The presence of progesterone receptor (PR) in estrogen receptor (ER)-positive breast cancer is associated with a good prognosis, and indicates that tumors are likely to respond to tamoxifen. However, ER+/PR− tumors respond less well. To reveal the potential molecular mechanism of this phenomenon, we sought to identify differential protein abundances between invasive ductal carcinoma cells from cryopreserved ER+/PR+ and ER+/PR− mammary tumor specimens. Because current proteomics methods are hampered in the examination of most primary human tumor samples by the extreme tissue heterogeneity, we used laser capture microdissection (LCM) to isolate tumor cells and developed a sample pooling strategy to analyze small sample protein lysates. Proteins from LCM-harvested tumors were pooled into four sub-pools from each condition of three tumors/sub-pool, and proteins from respective paired sub-pools were co-electrophoresed by 2-DE using 54-cm IEF over pH 4–9. Abundance ratios were accurately quantified by a differential multiplex radioactive ProteoTope method at low attomole levels (∼3.6 &μ;g protein per labeling reaction, <180 ng per multiplex protein sample per 54-cm gel). Applying this approach, differentially displayed proteins were identified by MS using comigrating non-radioactively labeled tumor proteins. They include decreased cytochrome b5 and transgelin, and more abundant CRABP-II, cyclophilin A, Neudesin, and hemoglobin in ER+/PR+ tumors versus ER+/PR−providing a possible explanation for differential susceptibility against tamoxifen as a result of deregulated cytochrome b5-dependent metabolism. This study demonstrates the potential of ProteoTope and LCM to enable extremely sensitive and precise differential analyses from well-defined primary clinical specimen.

- by Wojciech Wozny
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- Chemical Engineering, Analytical Chemistry, Breast Cancer, Proteomics
- by Eastwood Leung
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- Brain, Lasers, Laser Capture Microdissection, Microdissection

To identify specific markers of rectovaginal endometriotic nodule vasculature, highly enriched preparations of vascular endothelial cells and pericytes were obtained from endometriotic nodules and control endometrial and myometrial tissue by laser capture microdissection (LCM), and gene expression profiles were screened by microarray analysis. Of the 18 400 transcripts on the arrays, 734 were significantly overexpressed in vessels from fibromuscular tissue and 923 in vessels from stromal tissue of endometriotic nodules, compared with vessels dissected from control tissues. The most frequently expressed transcripts included known endothelial cell-associated genes, as well as transcripts with little or no previous association with vascular cells. The higher expression in blood vessels was further corroborated by immunohistochemical staining of six potential markers, five of which showed strong expression in pericytes. The most promising marker was matrix Gla protein, which was found to be present in both glandular epithelial cells and vascular endothelial cells of endometriotic lesions, although it was barely expressed at all in normal endometrium. LCM, combined with microarray analysis, constitutes a powerful tool for mapping the transcriptome of vascular cells. After immunohistochemical validation, markers of vascular endothelial and perivascular cells from endometriotic nodules could be identified, which may provide targets to improve early diagnosis or to selectively deliver therapeutic agents.

Although it is increasingly evident that cancer is influenced by signals emanating from tumor stroma, little is known regarding how changes in stromal gene expression affect epithelial tumor progression. We used laser capture microdissection to compare gene expression profiles of tumor stroma from 53 primary breast tumors and derived signatures strongly associated with clinical outcome. We present a new stroma-derived prognostic predictor (SDPP) that stratifies disease outcome independently of standard clinical prognostic factors and published expression-based predictors. The SDPP predicts outcome in several published whole tumor-derived expression data sets, identifies poor-outcome individuals from multiple clinical subtypes, including lymph node-negative tumors, and shows increased accuracy with respect to previously published predictors, especially for HER2-positive tumors. Prognostic power increases substantially when the predictor is combined with existing outcome predictors. Genes represented in the SDPP reveal the strong prognostic capacity of differential immune responses as well as angiogenic and hypoxic responses, highlighting the importance of stromal biology in tumor progression.

- by Margarita Souleimanova
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- Immune response, Breast Cancer, Gene expression, Lasers

Little is known about lung carcinoma epidermal growth factor (EGF) kinase pathway signaling within the context of the tissue microenvironment. We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within laser capture microdissected untreated, human non-small cell lung cancer (NSCLC) (n = 25) of known epidermal growth factor receptor (EGFR) tyrosine kinase domain mutation status. We measured six phosphorylation sites on EGFR to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR and downstream proteins. Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Tyr-1148 (p < 0.044) and Tyr-1068 (p < 0.026) and decreased phosphorylation of EGFR Tyr-1045 (p < 0.002), HER2 Tyr-1248 (p < 0.015), IRS-1 Ser-612 (p < 0.001), and SMAD Ser-465/467 (p < 0.011...

- by Claudia Fredolini and +2
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- Multidisciplinary, Signal Transduction, Cell line, Molecular and cellular biology
- by Gene Siegal
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- RNA, Science, Cryopreservation, Polymers

is an autosomal recessive disorder defined clinically by severe gastrointestinal dysmotility; cachexia; ptosis, ophthalmoparesis, or both; peripheral neuropathy; leukoencephalopathy; and mitochondrial abnormalities. The disease is caused by mutations in the thymidine phosphorylase (TP) gene. TP protein catalyzes phosphorolysis of thymidine to thymine and deoxyribose 1-phosphate. We identified 21 probands (35 patients) who fulfilled our clinical criteria for MNGIE. MNGIE has clinically homogeneous features but varies in age at onset and rate of progression. Gastrointestinal dysmotility is the most prominent manifestation, with recurrent diarrhea, borborygmi, and intestinal pseudo-obstruction. Patients usually die in early adulthood (mean, 37.6 years; range, 26 -58 years). Cerebral leukodystrophy is characteristic. Mitochondrial DNA (mtDNA) has depletion, multiple deletions, or both. We have identified 16 TP mutations. Homozygous or compound heterozygous mutations were present in all patients tested. Leukocyte TP activity was reduced drastically in all patients tested, 0.009 ؎ 0.021 mol/hr/mg (mean ؎ SD; n ‫؍‬ 16), compared with controls, 0.67 ؎ 0.21 mol/hr/mg (n ‫؍‬ 19). MNGIE is a recognizable clinical syndrome caused by mutations in thymidine phosphorylase. Severe reduction of TP activity in leukocytes is diagnostic. Altered mitochondrial nucleoside and nucleotide pools may impair mtDNA replication, repair, or both.

- by Antonella Spinazzola
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- Genetics, Cognitive Science, Neurology, Gastroenterology

Background: Esophageal squamous cell carcinomas (ESCC) are usually asymptomatic and go undetected until they are incurable. Cytological screening is one strategy to detect ESCC at an early stage and has shown promise in previous studies, although improvement in sensitivity and specificity are needed. Proteases modulate cancer progression by facilitating tumor invasion and metastasis. In the current study, matrix metalloproteinases (MMPs) were studied in a search for new early detection markers for ESCC. Methods: Protein expression levels of MMPs were measured using zymography in 24 cases of paired normal esophagus and ESCC, and in the tumor-associated stroma and tumor epithelium in one sample after laser capture microdissection (LCM). MMP-3 and MMP-10 transcripts in both the epithelium and stroma in five cases were further analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).

Context: Papillary thyroid carcinoma (PTC) is frequently multifocal. Independent PTC foci may occur either from intraglandular metastases from a single dominant tumor or as unrelated neoplastic clones. In rare cases, the simultaneous... more

- by Riccardo Giannini
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- Polymorphism, Mutation, Laser Capture Microdissection, Clinical

The multistage model of nonmelanoma skin carcinogenesis has contributed significantly to our understanding of epithelial cancer in general. We used the Krt1-15CrePR1;R26R transgenic mouse to determine the contribution of keratin 15þ cells from the hair follicle to skin tumor development by following the labeled progeny of the keratin 15 expressing cells into papillomas. We present three novel observations. First, we found that keratin 15 expressing cells contribute to most of the papillomas by 20 weeks of promotion. Second, in contrast to the transient behavior of labeled keratin 15-derived progeny in skin wound healing, keratin 15 progeny persist in papillomas, and some malignancies for many months following transient induction of the reporter gene. Third, papillomas have surprising heterogeneity not only in their cellular composition, but also in their expression of the codon 61 signature Ha-ras mutation with approximately 30% of keratin 15-derived regions expressing the mutation. Together, these results demonstrate that keratin 15 expressing cells of the hair follicle contribute to cutaneous papillomas with long term persistence and a subset of which express the Ha-ras signature mutation characteristic of initiated cells.

- by Derek Gordon
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- Stem Cells, Transgenic Mice, Keratinocytes, Mutation
- by Suel-Kee Kim
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- Gastroenterology, Immunohistochemistry, Cell separation, Cell Differentiation

The discovery of activating mutations in EGFR and KRAS in a subset of lung adenocarcinomas was a major advance in our understanding of lung adenocarcinoma biology, and has led to groundbreaking studies that have demonstrated the efficacy of tyrosine kinase inhibitor therapy. Fine-needle aspirates and other cytologic procedures have become increasingly popular for obtaining diagnostic material in lung carcinomas. However, frequently the small amount of material or sparseness of tumor cells obtained from cytologic preparations limit the number of specialized studies, such as mutation analysis, that can be performed. In this study we used laser capture microdissection to isolate small numbers of tumor cells to assess for EGFR and KRAS mutations from cell block sections of 19 cytology samples from patients with known lung adenocarcinomas. We compared our results with previous molecular assays that had been performed on either surgical or cytology specimens as part of the patient's initial clinical work-up. Not only were we able to detect the identical EGFR or KRAS mutation that was present in the patient's prior molecular assay in every case, but we were also able to consistently detect the mutation from as few as 50 microdissected tumor cells. Furthermore, isolating a more pure population of tumor cells resulted in increased sensitivity of mutation detection as we were able to detect mutations from laser capture microdissection-enriched cases where the tumor load was low and traditional methods of whole slide scraping failed. Therefore, this method can not only significantly increase the number of lung adenocarcinoma patients that can be screened for EGFR and KRAS mutations, but can also facilitate the use of cytologic samples in the newly emerging field of molecular-based personalized therapies.

- by Michael Emmert-Buck
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- Histology, Clinical Biochemistry, Apoptosis, Clinical Chemistry
- by Verity Oliver
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- RNA, Quality Control, Laser Capture Microdissection, Mice

Laser capture microdissection (LCM) is a well-established cell separation technique. It combines microscopy with laser beam technology and allows targeting of specific cells or tissue regions that need to be separated from others. Consequently, this biological material can be used for genome or transcriptome analyses. Appropriate methods of sample preparation, however, are crucial for the success of downstream molecular analysis. The aim of this study was to objectively compare the two main LCM systems, one based on an ultraviolet (UV) laser and the other based on an infrared (IR) laser, on different criteria ranging from user-friendliness to sample quality. The comparison was performed on two types of samples: peripheral blood mononuclear cells and blastocysts. The UV laser LCM system had several advantages over the IR laser LCM system. Not only does the UV system allow faster and more precise sample collection, but also the obtained samples-even single cell samples-can be used for DNA extraction and downstream polymerase chain reaction (PCR) applications. RNA-based applications are more challenging for both LCM systems. Although sufficient RNA can be extracted from as few as 10 cells for reverse transcription quantitative PCR (RT-qPCR) analysis, the low RNA quality should be taken into account when designing the RT-qPCR assays.

- by Dieter Deforce
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- Analytical Chemistry, RNA, DNA, Lasers
- by Sanjeev Sethi
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- Mass Spectrometry, Kidney diseases, Kidney, Laser Capture Microdissection

■ Abstract Aging is associated with deteriorated sinoatrial (SA) node function. The pacemaker current (I f ) carried by hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels plays a key role in the generation of spontaneous activity of the SA node cells. In the present study, the SA node cells were identified and isolated using the laser capture microdissection (LCM) technique for quantitative analysis of the HCN channel isoforms HCN1-HCN4 transcripts. Using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR), marked down-regulated transcriptions of HCN2 and HCN4 were observed in the SA node from young (1-month-old) to adult (4-month-old) and further to aged (30month-old) rats. However, neither the HCN1 nor HCN3 transcript was detectable throughout the lifespan of the rat. Consistently, the effect of 2 mM Cs + to selectively block the HCN channels, on pacemaking was also lessened with age. Our findings raise the possibility that the down-regulated transcription and relative function of HCN channels may contribute to the decline of the SA node function in aged rats.

- by A. Ma and +1
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- Aging, Ion Channels, Quantitative analysis, Laser Capture Microdissection

The age-dependent accumulation of point mutations in the control region of human mtDNA has been suggested to contribute to aging processes. We investigated whether mtDNA point mutations accumulate to detectable levels in this region of mtDNA from aged Fischer 344 X Brown Norway F 1 hybrid rats. The control region and a portion of the major arc region (nucleotides 4386-7707) of the mtDNA were PCRamplified and directly sequenced from microdissected single cardiomyocytes and single skeletal muscle fibers of 36-month old rats. Point mutations were not observed in these regions of the full-length mtDNA. Point mutations were, however, associated with deletion mutations, especially in cardiac cells. Approximately 40% of the deletion mutations identified in heart contained a point mutation, whereas only 1.9% of deletion mutations in skeletal muscle contained a point mutation. Point mutations were located adjacent to the deletion breakpoints and each point mutation was unique. In aged rats, point mutations are clonally expanded only when associated with deletion events suggesting that there are important differences between rats and humans in the mechanisms that cause mtDNA abnormalities.

- by Chad Johnson
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- Aging, Biology, Medicine, Mitochondrial DNA
- by Jason Theis
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- Nephrology, Urology, Mass Spectrometry, Kidney transplantation

We have used the mdx mice strain (C57BL/10ScSn-mdx) as an experimental subject for the study of reiterative skeletal muscle necrosis-regeneration with basement membrane preservation. In young mdx muscle, by means of Hematoxylin-Eosin staining, different types of degenerative-regenerative groups (DRG) can be recognized and assigned to a defined muscle regeneration phase. To evaluate the expression of known key-regulatory genes in muscle regeneration, we have applied Laser Capture Microdissection technique to obtain tissue from different DRGs encompassing the complete skeletal muscle regenerative process. The expression of MyoD, Myf-5 and Myogenin showed a rapid increase in the first two days post-necrosis, which were followed by MRF4 expression, when newly regenerating fibers started to appear (3-5 days post-necrosis). MHCd mRNA levels, undetectable in mature non-injured fibers, increased progressively from the first day post-necrosis and reached its maximum level of expression in DRGs showing basophilic regenerating fibers. TGFb-1 mRNA expression showed a prompt and strong increase following fiber necrosis that persisted during the inflammatory phase, and progressively decreased when new regenerating fibers began to appear. In contrast, IGF-2 mRNA expression decreased during the first days post-necrosis but was followed by a progressive rise in its expression coinciding with the appearance of the newly formed myofibers, reaching the maximum expression levels in DRGs composed of medium caliber basophilic regenerating myofibers (5-7 days post-necrosis). mdx degenerative-regenerative group typing, in conjunction with laser microdissection-based gene expression analysis, opens up a new approach to the molecular study of skeletal muscle regeneration.

- by Mario Marotta
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- Regeneration, Skeletal muscle biology, Basement Membrane, Lasers

Background: The Gene Ontology (GO) Consortium organizes genes into hierarchical categories based on biological process, molecular function and subcellular localization. Tools such as GoMiner can leverage GO to perform ontological analysis of microarray and proteomics studies, typically generating a list of significant functional categories. Two or more of the categories are often redundant, in the sense that identical or nearlyidentical sets of genes map to the categories. The redundancy might typically inflate the report of significant categories by a factor of three-fold, create an illusion of an overly long list of significant categories, and obscure the relevant biological interpretation.

- by Ari Kahn
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- Algorithms, Data Mining, Computational Biology, Proteomics

Rationale: Platelet-derived growth factor (PDGF) promotes the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), and may play a role in the progression of pulmonary arterial hypertension (PAH), a condition characterized by proliferation of PASMCs resulting in the obstruction of small pulmonary arteries. Objectives: To analyze the expression and pathogenic role of PDGF in idiopathic PAH. Methods: PDGF and PDGF receptor mRNA expression was studied by real-time reverse transcription-polymerase chain reaction performed on laser capture microdissected pulmonary arteries from patients undergoing lung transplantation for idiopathic PAH. Immunohistochemistry was used to localize PDGF, PDGF receptors, and phosphorylated PDGFR-b. The effects of imatinib on PDGF-B-induced proliferation and chemotaxis were tested on human PASMCs. Measurements and Main Results: PDGF-A, PDGF-B, PDGFR-a, and PDGFR-b mRNA expression was increased in small pulmonary arteries from patients displaying idiopathic PAH, as compared with control subjects. Western blot analysis revealed a significant increase in protein expression of PDGFR-b in PAH lungs, as compared with control lungs. In small remodeled pulmonary arteries, PDGF-A and PDGF-B mainly localized to PASMCs and endothelial cells (perivascular inflammatory infiltrates, when present, showed intensive staining), PDGFR-a and PDGFR-b mainly stained PASMCs and to a lesser extent endothelial cells. Proliferating pulmonary vascular lesions stained phosphorylated PDGFR-b. PDGF-BB-induced proliferation and migration of PASMCs were inhibited by imatinib. This effect was not due to PASMC apoptosis. Conclusions: PDGF may play an important role in human PAH. Novel therapeutic strategies targeting the PDGF pathway should be tested in clinical trials.

- by Rogério Souza
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- Neurosurgery, Clinical Trial, Apoptosis, Linear models

- by F. Pepin
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- Immune response, Breast Cancer, Gene expression, Lasers

Conventional molecular biology techniques enable gene expression analysis in a wide variety of analytical approaches and
paradigms. Experimental methods include Southern analysis (DNA detection), Northern analysis (RNA detection), polymerasechain
reaction (PCR; DNA detection), reverse-transcriptase-PCR (RT-PCR; RNA detection), ribonuclease (RNase) protection assay,
and in situ hybridization, among others. Newer next-generation sequencing approaches enable the assessment of noncoding RNAs
(ncRNAs) including microRNAs (miRNAs) and other regulatory RNA species. The majority of these conventional approaches
typically quantitate the abundance of individual elements one at a time (or a few at a time). Developments in high-throughput
genomic methodologies enable the assessment of dozens to hundreds to thousands of genes and ncRNAs simultaneously in a
coordinated manner. Recent advances in molecular genetics have led to sequencing of the entire human genome, as well as whole
genome sequencing of several relevant animal models. Furthermore, expression data is increasingly available for many diverse
tissues throughout the body, allowing for exciting hypothesis testing of critical concepts such as development, differentiation,
normative function, and ultimately, disease pathogenesis, which have translational potential.

- by Stephen D Ginsberg
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- Transcriptomics, Laser Capture Microdissection, Microarrays, RNA amplification
- by mark kockx
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- Genetics, Pathology, Molecular Biology, Cancer

The proliferation of keratinocytes in the hair follicle varies from slowly cycling, intermittently proliferating stem cells in the bulge to rapidly proliferating, transient cells in the bulb. To better understand the biological differences between these two compartments, we sought to identify differentially expressed genes using cDNA macroarray analysis. Cyclin D1 was one of 13 genes increased in the bulge compared to the bulb, and its differential expression was corroborated by quantitative real-time polymerase chain reaction (PCR) on the original samples. Using immunohistochemical staining, laser-capture microdissection (LCM) and quantitative real-time PCR, we localized cyclin D1 to the suprabasal cells of the telogen bulge and anagen outer root sheath (ORS). Surprisingly, cyclin D1, D2, and D3 were not detectable by immunohistochemistry in the rapidly proliferating hairproducing cells of the anagen bulb (matrix cells), while these cells were strongly positive for Ki-67 and retinoblastoma protein. In contrast, pilomatricoma, a tumor thought to be derived from matrix cells, was positive for cyclin D1, D2, and D3. Our results suggest that cyclin D1 may mediate the proliferation of stem cells in the bulge to more differentiated transient amplifying cells in the suprabasal ORS. In contrast, noncyclin D1-proteins appear to control cell division of the highly proliferative bulb matrix cells. This noncyclin D1-mediated proliferation may provide a protective mechanism against tumorigenesis, which is overridden in pilomatricomas. Our data also demonstrate that the combination of DNA macroarray, LCM and quantitative real-time PCR is a powerful approach for the study of gene expression in defined cell populations with limited starting material.

- by George Cotsarelis
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- Molecular Biology, Biology, Cell Cycle, Immunohistochemistry

The concept of tissues appeared more than 200 years ago, since textures and attendant differences were described within the whole organism components. Instrumental developments in optics and biochemistry subsequently paved the way to transition from classical to molecular histology in order to decipher the molecular contexts associated with physiological or pathological development or function of a tissue. In 1941, Coons and colleagues performed the first systematic integrated examination of classical histology and biochemistry when his team localized pneumonia antigens in infected tissue sections. Most recently, in the early 21 st century, mass spectrometry (MS) has progressively become one of the most valuable tools to analyze biomolecular compounds. Currently, sampling methods, biochemical procedures, and MS instrumentations allow scientists to perform ''in depth'' analysis of the protein content of any type of tissue of interest. This article reviews the salient issues in proteomics analysis of tissues. We first outline technical and analytical considerations for sampling and biochemical processing of tissues and subsequently the instrumental possibilities for proteomics analysis such as shotgun proteomics in an anatomical context. Specific attention concerns formalin fixed and paraffin embedded (FFPE) tissues that are potential ''gold mines'' for histopathological investigations. In all, the matrix assisted laser desorption/ionization (MALDI) MS imaging, which allows for differential mapping of hundreds of compounds on a tissue section, is currently the most striking evidence of linkage and transition between ''classical'' and ''molecular'' histology. Tissue proteomics represents a veritable field of research and investment activity for modern biomarker discovery and development for the next decade.

- by Nicolas Smargiasso
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- Proteomics, Histology, Immunohistochemistry, Biological Sciences
- by Dafna Benayahu
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- Genetics, RNA, Data Analysis, Carbon

Laser-assisted microdissection (LMD) has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with real-time PCR and RT-PCR techniques, it is now feasible to study genetic alterations, gene expression features and proteins in defined cell populations from complex normal and diseased tissues. The process that brings from sample collection to the final quantitative results is articulated in several steps, each of which requires optimal choices in order to end up with high-quality nucleic acid or protein that allows successful application of the final quantitative assays. This review will describe shortly the development of LMD technologies and the principles they are based on. Trying to highlight the advantages and disadvantages of LMD, the main problems related to specimens collection and processing, section preparation and extraction of biomolecules from microdissected tissue samples have been analysed.

- by Pamela Pinzani
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- Genetics, Gene expression, Sample Preparation, Biological Sciences

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°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.

- by Jack Su
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- Brain, Lasers, Laser Capture Microdissection, Microdissection

Background: Clinical and histological parameters are valid prognostic markers in renal disease, although they may show considerable interindividual variability and sometimes limited prognostic value. Novel molecular markers and pathways have the potential to increase the predictive prognostic value of the so called "traditional markers". Methods: Transcriptomics profiles from laser-capture microdissected proximal tubular epithelial cells from routine kidney biopsies were correlated with a chronic renal damage index score (CREDI), an inflammation score (INSCO), and clinical parameters. We used data from 20 renal biopsies with various proteinuric renal diseases with a median follow-up of 49 months (discovery cohort). For validation we performed microarrays from whole kidney biopsies from a second cohort consisting of 16 patients with a median follow-up time of 28 months (validation cohort).

- by Gert Mayer
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- Genetics, Inflammation, Laser Capture Microdissection, Human Genome

Several studies have demonstrated that the mammalian retina contains an autonomous circadian clock. Dopaminergic and other inner retinal neurons express many of the clock genes, whereas some of these genes seem to be absent from the photoreceptors. This observation has led to the suggestion that in mammalian retina the circadian pacemaker driving retinal rhythms is located in the inner nuclear layer. However, other evidence points to the photoreceptor layer as the site of the mammalian retinal clock. The goal of the present study was to demonstrate the presence of a functional circadian clock in photoreceptors. First, using laser capture microdissection and reverse transcriptase-polymerase chain reaction, we investigated which of the clock genes are expressed in rat photoreceptors. We then prepared photoreceptor layer cultures from the retina to test whether these isolated cultures were viable and could drive circadian rhythms. Our data indicated that Per1, Per3, Cry1, Cry2, Clock, Bmal1, Rev-erb␣, and Rora RNAs were present in the photoreceptors, whereas we were unable to amplify mRNA for Per2 and Npas2. Photoreceptor layers obtained from Period1-luciferase rats expressed a robust circadian rhythm in bioluminescence and melatonin synthesis. These results demonstrate that mammalian photoreceptors contain the circadian pacemaker driving rhythmic melatonin synthesis.-Tosini, G., Davidson, A. J., Fukuhara, C., Kasamatsu, M., Castanon-Cervantes, O. Localization of a circadian clock in mammalian photoreceptors. FASEB J. 21, 3866 -3871 (2007)

- by O. Castanon-cervantes
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- Physiology, Melatonin, Laser Capture Microdissection, Circadian Rhythm

We have studied hypertrophic and immediately adjacent pre-hypertrophic chondrocytes at the same stage of histologic development in 7 day old post-natal Balb/C mouse physes and epiphyses. Laser capture microdissection (LCM) and GeneChip microarray analysis compared the molecular composition of the two hypertrophic chondrocyte regions. Molecules upregulated in dramatically higher levels in the epiphysis were gremlin (58-fold), epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (25-fold), and frizzled related protein (6.4-fold and 5.7-fold). Molecules upregulated in higher levels in the physis were proline arginine-rich end leucine-rich repeat protein (PRELP) (15.6-fold), pyrophosphatase (inorganic) 1 (10-fold) and hedgehog-interacting protein (7.3-fold). Immunocytochemistry for gremlin confirmed specific localization patterns. This study indicates a critical site-specific role for hypertrophic chondrocytes with different synthesis patterns in separate regions even though they appear structurally the same and are at the same stage of development.

- by Monica Calicchio
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- Cell Differentiation, Microarray, Laser Capture Microdissection, Mice

It is well known that sex steroids are involved in the growth of breast cancers, and the great majority of breast carcinomas express estrogen (ER), progesterone (PR), and androgen (AR) receptors. In particular, recent studies have demonstrated that estrogens and androgens are locally produced in breast carcinoma tissues, and total blockade of in situ estrogen production potentially leads to an improvement in prognosis of breast cancer patients. Therefore, it is important to obtain a better understanding of sex steroid-producing enzymes in breast carcinoma tissues. In this review, we summarize recent studies on the expression and regulation of enzymes related to intratumoral production of estrogens (aromatase, 17b-hydroxysteroid dehydrogenase type 1 (17bHSD1), and steroid sulfatase (STS) etc) and androgens (17bHSD5 and 5a-reductase) in human breast carcinoma tissues, and discuss the biological and/or clinical significance of these enzymes. The cellular localization of aromatase in breast carcinoma tissues still remains controversial. Therefore, we examined localization of aromatase mRNA in breast carcinoma tissues by laser capture microdissection/real time-polymerase chain reaction. Aromatase mRNA expression was detected in both carcinoma and intratumoral stromal cells, and the expression level of aromatase mRNA was higher in intratumoral stromal cells than in carcinoma cells in the cases examined. We also examined an association among the immunoreactivity of enzymes related to intratumoral estrogen production and ERs in breast carcinoma tissues, but no significant association was detected. Therefore, the enzymes responsible for the intratumoral production of estrogen may not always be the same among breast cancer patients, and not only aromatase but also other enzymes such as STS and 17bHSD1 may have important therapeutic potential as targets for endocrine therapy in breast cancer patients.

- by Noriaki Ohuchi and +1
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- Breast Cancer, Enzyme Inhibitors, Biological Sciences, Laser Capture Microdissection

While the clinical and neuropathological characterization of Alzheimer's Disease (AD) is well defined, our understanding of the progression of pathologic mechanisms in AD remains unclear. Post-mortem brains from individuals who did not fulfill clinical criteria for AD may still demonstrate measurable levels of AD pathologies to suggest that they may have presented with clinical symptoms had they lived longer or are able to stave off disease progression. Comparison between such individuals and those clinically diagnosed and pathologically confirmed to have AD will be key in delineating AD pathogenesis and neuroprotection. In this study, we expression profiled laser capture microdissected non-tangle bearing neurons in 6 post-mortem brain regions that are differentially affected in the AD brain from 10 non-demented individuals demonstrating intermediate AD neuropathologies (NDAD; Braak stage of II through IV and CERAD rating of moderate to frequent) and evaluated this data against that from individuals who have been diagnosed with late onset AD as well as healthy elderly controls. We identified common statistically significant expression changes in both NDAD and AD brains that may establish a degenerative link between the two cohorts, in addition to NDAD specific transcriptomic changes. These findings pinpoint novel targets for developing earlier diagnostics and preventative therapies for AD prior to diagnosis of probable AD. We also provide this high-quality, low post-mortem interval (PMI), cell-specific, and region-specific NDAD/AD reference data set to the community as a public resource.

- by Walter Kukull
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- Transcriptomics, Neurobiology, Gene expression, Brain

Soybean is one of the top five agricultural products in the United States. Soybean rust is caused by the obligate fungus Phakopsora pachyrhizi Sydow, an exotic pathogen in the US Extensive screening of soybean germplasm has not identified... more

- by Benjamin Matthews
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- Microbiology, Plant Biology, Gene expression, Signal Transduction

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), a-tubulin (GmTubA1), b-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.

- by Benjamin Matthews
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- Genetics, RNA, Plant Biology, Plant Molecular Biology
- by Giacomo Accardo and +1
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- Obstetrics, Regulation, Laser Capture Microdissection, Microdissection
- by Wesley Garrett
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- Microbiology, Immune response, Immunohistochemistry, Veterinary

Melanoma may be difficult to identify histologically and relatively high rates of misdiagnosis leads to many malpractice claims. Currently separation of melanomas from nevi is based primarily on light microscopic interpretation of hematoxylin and eosin stained sections with limited assistance from immunohistology. To increase the accuracy of discrimination of benign and malignant melanocytic lesions we identified DNA microarray-derived gene expression profiles of different melanocytic lesions and evaluated the performance of these gene signatures as molecular diagnostic tools in the molecular classification and separation of melanomas and nevi. Melanocyte-derived cells were isolated by laser capture microdissection from 165 formalin-fixed and paraffin-embedded melanocytic nevi and melanoma tissue sections. RNA was isolated, amplified, labeled, and hybridized to a custom DNA microarray. In all 120 samples were used to identify differentially expressed genes and generate a gene expression classifier capable of distinguishing between melanomas and nevi. These classifiers were tested by the leave-one-out method and in a blinded study. RT-PCR verified the results. Unsupervised hierarchical clustering identified two distinct lesional groups that closely correlated with the histopathologically identified melanomas and nevi. Analysis of gene expression levels identified 36 significant differentially expressed genes. In comparison with nevi, melanomas expressed higher levels of genes promoting signal transduction, transcription, and cell growth. In contrast, expression of L1CAM (homolog) was reduced in melanomas relative to nevi. Genes differentially expressed in melanomas and nevi, on the basis of molecular signal, sub classified a group of unknown melanocytic lesions as melanomas or nevi and had high concordance rates with histopathology. Gene signatures established using DNA microarray gene expression profiling can distinguish melanomas from nevi, indicating the feasibility of using molecular classification as a supplement to standard histology. Our successful use of a standard formalin-fixed and paraffin-embedded tissue further supports the practicability of combining molecular diagnostic testing with histopathology in evaluation of difficult melanocytic lesions.

- by Eric Whitman
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- Histology, Clinical Biochemistry, Apoptosis, Gene expression

Described herein is a detailed analysis of the impact of three fixatives (10% neutral buffered formalin, modified methacarn and 70% ethanol) on RNA quality and utility using microarray analysis compared to OCT-embedded and flash frozen tissue. From rat livers fixed and stored in paraffin blocks for 1 month or 1 year, RNA was isolated and applied to rat whole genome microarrays. At both time points, RNA isolated from OCT-embedded tissue lost up to 5% of the information contained in snap frozen control liver. Of the fixatives used, modified methacarn was associated with the smallest loss of RNA information content (approximately 10%), while liver fixed in 70% ethanol and 10% neutral buffered formalin lost roughly 25% and 80%, respectively. We conclude that when optimum morphology is required for techniques such as laser microdissection, modified methacarn is the fixative least harmful to nucleic acids of the three tested in this study. In contrast, using traditional isolation techniques, RNA derived from tissue fixed in 10% NBF will not give reliable results on microarray studies, and should be reserved for techniques less affected by the fragmentation and modification of the template RNA, such as quantitative RT-PCR.

- by Melissa L. Cox
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- Microarray, Laser Capture Microdissection, Fixatives, Tissue Fixation
- by Maciej Lalowski
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- Biology, Proteomics, Mitochondria, Medicine

Annexin I protein expression was evaluated in patient-matched longitudinal study sets of laser capture microdissected normal, premalignant, and invasive epithelium from human esophageal squamous cell cancer and prostatic adenocarcinoma. In 25 esophageal cases (20 by Western blot and 5 by immunohistochemistry) and 17 prostate cases (3 by Western blot and 14 by immunohistochemistry), both tumor types showed either complete loss or a dramatic reduction in the level of annexin I protein expression compared with patient-matched normal epithelium (P < 0.05). Moreover, by using Western blot analysis of laser capture microdissected, patient-matched longitudinal study sets of both tumor types, the loss of protein expression occurred in premalignant lesions. Concordance of this result with immunohistochemical analysis suggests that annexin I may be an essential component for maintenance of the normal epithelial phenotype. Additional studies investigating the mechanism(s) and functional consequences of annexin I protein loss in tumor cells are warranted.

- by Judi Herring
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- Cancer, Immunohistochemistry, Western blotting, Laser Capture Microdissection
- by Vipin Yadav
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- Cellular Biology, Molecular Biology, Skin Cancer, Laser Capture Microdissection

METHODS: Leukocyte adherence, emigration and permeability to FITC-albumin were measured in mesenteric venules of anesthetized rats using intravital microscopy. Rats were acclimatized to hypobaric Hx for 3 weeks at a level equivalent to 10% inspired O2. Hematocrit of acclimatized rats was reduced to normal levels before experiments. Tissue HO protein was measured by Western blots. Responses in non-acclimatized (NA) and acclimatized (AC) rats were compared. Zinc protoporphyrin (ZnPP) was administered to inhibit HO.

- by Christiane Ferran and +1
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- Laser Capture Microdissection, The, Clinical Sciences, Gene Expression Analysis

Vein graft failure occurs between 1 and 6 months after implantation due to obstructive intimal hyperplasia, related in part to implantation injury. The cell-specific and temporal response of the transcriptome to vein graft implantation injury was determined by transcriptional profiling of laser capture microdissected endothelial cells (EC) and medial smooth muscle cells (SMC) from canine vein grafts, 2 hours (H) to 30 days (D) following surgery. Our results demonstrate a robust genomic response beginning at 2 H, peaking at 12-24 H, declining by 7 D, and resolving by 30 D. Gene ontology and pathway analyses of differentially expressed genes indicated that implantation injury affects inflammatory and immune responses, apoptosis, mitosis, and extracellular matrix reorganization in both cell types. Through backpropagation an integrated network was built, starting with genes differentially expressed at 30 D, followed by adding upstream interactive genes from each prior time-point. This identified significant enrichment of IL-6, IL-8, NF-kB, dendritic cell maturation, glucocorticoid receptor, and Triggering Receptor Expressed on Myeloid Cells (TREM-1) signaling, as well as PPARa activation pathways in graft EC and SMC. Interactive network-based analyses identified IL-6, IL-8, IL-1a, and Insulin Receptor (INSR) as focus hub genes within these pathways. Real-time PCR was used for the validation of two of these genes: IL-6 and IL-8, in addition to Collagen 11A1 (COL11A1), a cornerstone of the backpropagation. In conclusion, these results establish causality relationships clarifying the pathogenesis of vein graft implantation injury, and identifying novel targets for its prevention.

- by Manoj Bhasin
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- Engineering, Physics, Chemistry, Immune response