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As the need for high-speed electronics continues to rise rapidly, printed wiring board (PWB) requirements become ever-more demanding. A typical PWB is fabricated by bonding dielectric films such as polyimide to electrically conductive copper foil such as rolled annealed (RA) copper and is expected to become thinner, flexible, durable, and compatible with high-frequency 5G performance. Polyimide films inherently feature a higher coefficient of thermal expansion (CTE) than copper foils; this mismatch causes residual thermal stresses. To attenuate the mismatch, silica nanoparticles may be used to reduce the CTE of PI. A nodulated copper surface can be used to enhance the Cu/PI adhesion by additional bonding mechanisms that could include a type of mechanical bonding, which is a focus of this study. In this investigation, a 90° peel test was used to measure the peel strength in copper/polyimide/copper laminates containing nodulated copper and polyimide reinforced with 0, 20, and 40 wt % silica nanoparticles. The influence of silica nanoparticles on the peel strength was quantitatively evaluated. Laminates incorporating polyimide films lacking silica nanoparticles had a â¼3.75× higher peel strength compared with laminates reinforced with 40% silica. Their failure surfaces were analyzed by using scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDX), and X-ray photoelectron spectroscopy to identify the mode of failure and to understand bonding mechanisms. The key bonding mechanism, mechanical interlocking, was achieved when the polyimide surrounded or engulfed the copper nodules when the laminate was created. Post-testing failure surface analysis revealed the presence of copper on the polyimide side and polyimide on the copper side, indicating mixed mode failure. An analytical model was developed to determine the impact of applied pressure, temperature, and time on the polyimide penetration and mechanical interlocking around the copper nodules. The model was validated by measuring the peel strength on another set of specimens fabricated using increased temperature and pressure that showed a 3× increase in peel strength compared to lower temperature/pressure processing conditions. This enhanced adhesion resulted from the lower polymer material viscosity at higher temperatures, which fosters deeper and more complete penetration around the copper nodules during processing at higher pressures for longer durations. The methodology of combining peel testing, viscosity and CTE measurement, SEM/EDX, surface chemical analysis, and penetration depth calculation developed herein enables the calculation of the desired processing parameters to enhance functionality and improve adhesion.
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Measurement of gene-expression profiles using microarray technology is becoming increasingly popular among the biomedical research community. Although there has been great progress in this field, investigators are still confronted with a difficult question after completing their experiments: how to validate the large data sets that are generated? This review summarizes current approaches to verifying global expression results, discusses the caveats that must be considered, and describes some methods that are being developed to address outstanding problems.
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Perfilación de la Expresión Génica/normas , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , Animales , ADN Complementario/genética , Predicción , Perfilación de la Expresión Génica/métodos , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas/genética , Control de Calidad , ARN Mensajero/genética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We address the dilemma faced by oncologists in administering preventative measures to "at risk" patients diagnosed with atypical and nonatypical hyperplasias due to lack of any molecular means of risk stratification and identifying high-risk subjects. Our study purpose is to investigate a four marker risk signature, MMP-1, CEACAM6, HYAL1, and HEC1, using 440 hyperplastic tissues for identifying high-risk subjects who will benefit from preventative therapies. We assayed the markers by IHC and combined their expression levels to obtain a composite value from 0-10, which we called a "Cancer Risk Score." We demonstrate that the four marker-based risk scores predict subsequent cancer development with an accuracy of 91% and 86% for atypical and nonatypical subjects, respectively. We have established a correlation between risk scores and cancer rates by stratifying the samples into low risk (score ≤ 0.5); intermediate risk (score ≤ 5.4), and high risk (score >5.4) groups using Kaplan-Meier survival analysis. We have evaluated cancer rates at 5, 10, and 15 years. Our results show that the average cancer rates in the first 5 years among low- and intermediate-risk groups were 2% and 15%, respectively. Among high-risk group, the average cancer rates at 5 years were 73% and 34% for atypical and nonatypical subjects, respectively. The molecular risk stratification described here assesses a patient's tumor biology-based risk level as low, intermediate, or high and for making informed treatment decisions. The outcomes of our study in conjunction with the available prophylactic measures could prevent approximately 20%-25% of sporadic breast cancers.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Carcinoma Lobular/patología , Hiperplasia/patología , Medición de Riesgo/métodos , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/epidemiología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/epidemiología , Carcinoma Lobular/metabolismo , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Humanos , Hiperplasia/epidemiología , Hiperplasia/metabolismo , Incidencia , Persona de Mediana Edad , Pronóstico , Factores de Riesgo , Estados Unidos/epidemiologíaRESUMEN
Procurement of pure populations of cells from heterogeneous histological sections can be accomplished utilizing tissue microdissection. At present, a variety of different manual and laser-based dissection tools are available and each method has particular strengths and weaknesses. The types of biomolecular analyses that can be performed on microdissected cells depend not only on the method of cell procurement, but also on the effects of upstream tissue handling and processing. Tissue preparation protocols include two major approaches; snap-freezing, or, fixation and embedding. Snap-freezing generally provides the best quality tissue for subsequent study, including proteomic analyses such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Tissue fixatives include either precipitating reagents or biomolecular cross-linkers. The fixed samples are then further processed and embedded in a wax medium. In general, the biomolecules recovered from fixed and embedded tissue specimens are lower in both quantity and quality than those from snap-frozen specimens, although they are useful for certain types of analyses. The protocols provided here for tissue handling and processing, preparation of tissue sections, and microdissection are derived from our experience at the Pathogenetics Unit of the National Cancer Institute.
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Criopreservación/métodos , Microdisección/instrumentación , Microdisección/métodos , Fijación del Tejido/métodos , Animales , Histocitoquímica/métodos , HumanosRESUMEN
Periocular necrotizing fasciitis is a rare, but potentially blinding, or even fatal disease. The authors report a case of a 44-year-old man who presented with quiescent bilateral periocular and facial necrotizing fasciitis. The patient was treated with antibiotics and surgical debridement, followed by negative-pressure wound therapy (NPWT), until the wound bed was thought to be healthy enough to support bilateral upper eyelid full-thickness skin grafts. NPWT appeared to decrease local edema; speed reperfusion and granulation tissue formation; and served to stabilize the skin grafts against the wound bed, while not causing any ocular complications. NPWT can be a safe and effective adjunct treatment for periocular necrotizing fasciitis.
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Layered peptide array is a new methodology for multiplex molecular measurements from two-dimensional life science platforms. The technology can be used in several different configurations depending on the needs of the investigator. Described here is an indirect layered peptide array (iLPA) that is capable of measuring proteins on a solid surface, such as target antigens on a tissue section. A prototype iLPA system was developed and subsequently examined for reproducibility and specificity and then compared with standard immunohistochemistry. Semiquantitative, multiplex proteomic analysis of histological sections was achieved with up to 20 membranes. The experimental variability was 18%. Overall, the data suggest that iLPA technology will be a relatively simple and inexpensive method for molecular measurements from tissue sections.
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Inmunohistoquímica/métodos , Fragmentos de Péptidos/análisis , Análisis por Matrices de Proteínas/métodos , Neoplasias Encefálicas/metabolismo , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Neoplasias del Colon/metabolismo , Estudios de Factibilidad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/metabolismo , Linfoma/metabolismo , Masculino , Melanoma/metabolismo , Modelos Biológicos , Neurilemoma/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias de la Próstata/metabolismo , Análisis de Matrices Tisulares/métodosRESUMEN
The layered peptide array (LPA) is a recently developed technique designed to measure antibody levels in a multiplex, high-throughput manner. LPAs can assess antibody presence either in fluid samples or from tissues while maintaining the two-dimensional orientation of the life science platform. In this manuscript, we evaluated and assessed the performance of the LPA platform, focusing on throughput capability, sensitivity, and specificity of the assay in several different systems.
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Anticuerpos/análisis , Péptidos/análisis , Análisis por Matrices de Proteínas , Anticuerpos/sangre , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Péptidos/sangreRESUMEN
BACKGROUND: A functional blood supply is essential for tumor growth and proliferation. However, the mechanism of blood vessel recruitment to the tumor is still poorly understood. Ideally, a thorough molecular assessment of blood vessel cells would be critical in our comprehension of this process. Yet, to date, there is little known about the molecular makeup of the endothelial cells of tumor-associated blood vessels, due in part to the difficulty of isolating a pure population of endothelial cells from the heterogeneous tissue environment. METHODS: Here we describe the use of a recently developed technique, Expression Microdissection, to isolate endothelial cells from the tumor microenvironment. The methylation status of the dissected samples was evaluated for GSTP1 and RARbeta2 promoters via the QMS-PCR method. RESULTS: Comparing GSTP1 and RARbeta2 promoter methylation data, we show that 100% and 88% methylation is detected, respectively, in the tumor areas, both in epithelium and endothelium. Little to no methylation is observed in non-tumor tissue areas. CONCLUSION: We applied an accurate microdissection technique to isolate endothelial cells from tissues, enabling DNA analysis such as promoter methylation status. The observations suggest that epigenetic alterations may play a role in determining the phenotype of tumor-associated vasculature.
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PURPOSE: After an initial response to androgen ablation, most prostate tumors recur, ultimately progressing to highly aggressive androgen-independent cancer. The molecular mechanisms underlying progression are not well known in part due to the rarity of androgen-independent samples from primary and metastatic sites. EXPERIMENTAL DESIGN: We compared the gene expression profiles of 10 androgen-independent primary prostate tumor biopsies with 10 primary, untreated androgen-dependent tumors. Samples were laser capture microdissected, the RNA was amplified, and gene expression was assessed using Affymetrix Human Genome U133A GeneChip. Differential expression was examined with principal component analysis, hierarchical clustering, and Student's t testing. Analysis of gene ontology was done with Expression Analysis Systematic Explorer and gene expression data were integrated with genomic alterations with Differential Gene Locus Mapping. RESULTS: Unsupervised principal component analysis showed that the androgen-dependent and androgen-independent tumors segregated from one another. After filtering the data, 239 differentially expressed genes were identified. Two main gene ontologies were found discordant between androgen-independent and androgen-dependent tumors: macromolecule biosynthesis was down-regulated and cell adhesion was up-regulated in androgen-independent tumors. Other differentially expressed genes were related to interleukin-6 signaling as well as angiogenesis, cell adhesion, apoptosis, oxidative stress, and hormone response. The Differential Gene Locus Mapping analysis identified nine regions of potential chromosomal deletion in the androgen-independent tumors, including 1p36, 3p21, 6p21, 8p21, 11p15, 11q12, 12q23, 16q12, and 16q21. CONCLUSIONS: Taken together, these data identify several unique characteristics of androgen-independent prostate cancer that may hold potential for the development of targeted therapeutic intervention.
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Andrógenos/metabolismo , Antineoplásicos Hormonales/farmacología , Regulación Neoplásica de la Expresión Génica , Regulación de la Expresión Génica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/terapia , Anciano , Antagonistas de Andrógenos/metabolismo , Biopsia , Adhesión Celular , Deleción Cromosómica , Mapeo Cromosómico , Cromosomas/ultraestructura , Análisis por Conglomerados , Progresión de la Enfermedad , Regulación hacia Abajo , Eliminación de Gen , Genoma , Humanos , Interleucina-6/metabolismo , Rayos Láser , Masculino , Persona de Mediana Edad , Modelos Estadísticos , Metástasis de la Neoplasia , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo , Análisis de Componente Principal , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN/metabolismo , Transducción de Señal , Regulación hacia ArribaRESUMEN
We analyzed expression status of the recently identified tumor suppressor geneRASSF1A in primary prostate carcinomas and in prostate cell lines. We found complete methylation of the RASSF1A promoter in 63% of primary microdissected prostate carcinomas (7 of 11 samples). The remaining 4 samples (37%) were partially methylated, possibly because of contamination with normal cells. No promoter methylation was observed in matching normal prostate tissues. High levels of RASSF1A transcript and no methylation of RASSF1A promoter were found in explanted primary normal prostate epithelial and stromal cells. Complete silencing and methylation of RASSF1A promoter was observed in five widely used prostate carcinoma cell lines, which acquired the ability to grow in culture spontaneously, including LNCaP, PC-3, ND-1, DU-145, 22Rv1, and one primary prostate carcinoma immortalized by overexpression of the human telomerase catalytic subunit (RC-58T/hTERT). However, no silencing of RASSF1A was found in four other prostate carcinoma cell lines, which were adapted for cell culture after transformation with human papillomaviral DNA. Suppression of cell growth in vitro was demonstrated after the reintroduction of RASSF1A-expressing construct into LNCaP prostate carcinoma cells. Our data implicate the RASSF1A gene in human prostate tumorigenesis.
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Silenciador del Gen , Genes Supresores de Tumor , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/genética , Proteínas Supresoras de Tumor , División Celular/genética , Transformación Celular Viral , Metilación de ADN , Proteínas de Unión al ADN , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Proteínas de Neoplasias/biosíntesis , Papillomaviridae/fisiología , Regiones Promotoras Genéticas , Próstata/citología , Próstata/metabolismo , Próstata/fisiología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Células del Estroma/metabolismo , Células del Estroma/fisiología , Telomerasa/genética , Células Tumorales CultivadasRESUMEN
The electrophoretic deposition (EPD) method was used to deposit polyethylenimine (PEI) functionalized multiwall carbon nanotube (CNT) films onto the surface of individual S-2 glass fibers. By varying the processing parameters of EPD following Hamaker's equation, the thickness of the CNT film was controlled over a wide range from 200 nm to 2 µm. The films exhibited low electrical resistance, providing evidence of coating uniformity and consolidation. The effect of the CNT coating on fiber matrix interfacial properties was investigated through microdroplet experiments. Changes in interfacial properties due to application of CNT coatings onto the fiber surface with and without a CNT-modified matrix were studied. A glass fiber with a 2 µm thick CNT coating and the unmodified epoxy matrix showed the highest increase (58%) in interfacial shear strength (IFSS) compared to the baseline. The increase in the IFSS was proportional to CNT film thickness. Failure analysis of the microdroplet specimens indicated higher IFSS was related to fracture morphologies with higher levels of surface roughness. EPD enables the thickness of the CNT coating to be adjusted, facilitating control of fiber/matrix interfacial resistivity. The electrical sensitivity provides the opportunity to fabricate a new class of sizing with tailored interfacial properties and the ability to detect damage initiation.
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Smokers develop metastatic prostate cancer more frequently than nonsmokers, suggesting that a tobacco-derived factor is driving metastatic progression. To identify smoking-induced alterations in human prostate cancer, we analyzed gene and protein expression patterns in tumors collected from current, past, and never smokers. By this route, we elucidated a distinct pattern of molecular alterations characterized by an immune and inflammation signature in tumors from current smokers that were either attenuated or absent in past and never smokers. Specifically, this signature included elevated immunoglobulin expression by tumor-infiltrating B cells, NF-κB activation, and increased chemokine expression. In an alternate approach to characterize smoking-induced oncogenic alterations, we also explored the effects of nicotine in human prostate cancer cells and prostate cancer-prone TRAMP mice. These investigations showed that nicotine increased glutamine consumption and invasiveness of cancer cells in vitro and accelerated metastatic progression in tumor-bearing TRAMP mice. Overall, our findings suggest that nicotine is sufficient to induce a phenotype resembling the epidemiology of smoking-associated prostate cancer progression, illuminating a novel candidate driver underlying metastatic prostate cancer in current smokers.
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Inflamación/metabolismo , Neoplasias de la Próstata/inmunología , Fumar/efectos adversos , Transcriptoma , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Inmunoglobulinas/genética , Interleucina-8/sangre , Masculino , Ratones , FN-kappa B/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Nicotina/farmacología , Neoplasias de la Próstata/etiología , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
High-throughput methods to detect and quantify antibodies in sera and other patient specimens have use for many clinical and laboratory studies, including those associated with cancer detection, microbial exposures, and autoimmune diseases. We developed a new technique, termed layered peptide array (LPA), to serve as a screening tool to detect antibodies in a highly multiplexed format. We demonstrate here that a prototype LPA was capable of producing approximately 5000 measurements per experiment and appeared to be scalable to higher throughput levels. Sera and saliva from Sjögren's syndrome patients served as a test set to examine antibody titers in clinical samples. The LPA platform exhibited both a high sensitivity (100%) and high specificity (94%) for correctly identifying SSB antigen-positive samples. The multiplex capability of the platform was also confirmed when serum and saliva samples were analyzed for antibody reactivity to several peptides, including Sjögren's syndrome antigens A and B. The data indicate that LPA analysis will be a useful method for a number of screening applications.
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Anticuerpos/análisis , Anticuerpos/inmunología , Tamizaje Masivo/métodos , Péptidos/inmunología , Análisis por Matrices de Proteínas/métodos , Análisis por Conglomerados , Humanos , Tamizaje Masivo/instrumentación , Péptidos/química , Análisis por Matrices de Proteínas/instrumentación , Saliva/inmunología , Síndrome de Sjögren/sangre , Síndrome de Sjögren/diagnóstico , Síndrome de Sjögren/inmunologíaRESUMEN
Proteomic analysis of clinical tissue specimens is a difficult undertaking. Described here is a multiplex study of protein expression levels in histological sections of human prostate that addresses many of the associated challenges. Whole-mount sections from 10 prostatectomy specimens were studied using 15 antibodies, immunohistochemical staining, digital imaging, and mathematical analysis of the data sets. The approach was successful in stratifying cell lineages present in the samples based on proteomic patterns, including differentiating normal epithelium from cancer. This strategy likely will be a useful method for extending the number of proteins that can be analyzed in clinical cancer specimens using currently available laboratory techniques.
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Próstata/metabolismo , Proteínas/metabolismo , Células Epiteliales/metabolismo , Epitelio/metabolismo , Epitelio/patología , Estudios de Factibilidad , Humanos , Inmunohistoquímica , Masculino , Matemática , Análisis de Componente Principal , Próstata/citología , Próstata/patología , Hiperplasia Prostática/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ProteómicaRESUMEN
Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.
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Separación Celular/métodos , Células del Tejido Conectivo/metabolismo , Perfilación de la Expresión Génica/métodos , Microdisección/métodos , Proteoma/metabolismo , Manejo de Especímenes/métodos , Conservación de Tejido/métodos , Biomarcadores/metabolismo , Células del Tejido Conectivo/clasificación , Biología Molecular/métodosRESUMEN
A number of genetic and epigenetic changes underlying the development of nasopharyngeal carcinomas have recently been identified. However, there is still limited information on the nature of the genes and gene products whose aberrant expression and activity promote the malignant conversion of nasopharyngeal epithelium. Here, we have performed a genome-wide transcriptome analysis by probing cDNA microarrays with fluorescent-labeled amplified RNA derived from laser capture microdissected cells procured from normal nasopharyngeal epithelium and areas of metaplasia-dysplasia and carcinoma from EBV-associated nasopharyngeal carcinomas. This approach enabled the identification of genes differentially expressed in each cell population, as well as numerous genes whose expression can help explain the aggressive clinical nature of this tumor type. For example, genes indicating cell cycle aberrations (cyclin D2, cyclin B1, activator of S-phase kinase, and the cell cycle checkpoint kinase, CHK1) and invasive-metastatic potential (matrix metalloproteinase 11, v-Ral, and integrin beta(4)) were highly expressed in tumor cells. In contrast, genes underexpressed in tumors included genes involved in apoptosis (B-cell CLL/lymphoma 6, secretory leukocyte protease inhibitor, and calpastatin), cell structure (keratin 7 and carcinoembryonic antigen-related cell adhesion molecule 6), and putative tumor suppressor genes (H-Ras-like suppressor 3, retinoic acid receptor responder 1, and growth arrested specific 8) among others. Gene expression patterns also suggested alterations in the Wnt/beta-catenin and transforming growth factor beta pathways in nasopharyngeal carcinoma. Thus, expression profiles indicate that aberrant expression of growth, survival, and invasion-promoting genes may contribute to the molecular pathogenesis of nasopharyngeal carcinoma. Ultimately, this approach may facilitate the identification of clinical useful markers of disease progression and novel potential therapeutic targets for nasopharyngeal carcinoma.
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Carcinoma/genética , Carcinoma/metabolismo , ADN/ultraestructura , Regulación Neoplásica de la Expresión Génica , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Línea Celular Tumoral , Cartilla de ADN/química , ADN Complementario/metabolismo , Progresión de la Enfermedad , Epitelio/patología , Genes Supresores de Tumor , Genoma , Humanos , Inmunohistoquímica , Rayos Láser , Neoplasias/metabolismo , Proteoma/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of pancreatic malignancy and other biological phenomena. This chapter reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed-over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification. High-quality tissue microdissection does not necessarily mean high-quality samples to analyze. The quality of biomaterials obtained for analysis is highly dependent on steps upstream and downstream from tissue microdissection. We provide protocols for each of these steps, and encourage you to improve upon these. It is worth the effort of every laboratory to optimize and document its technique at each stage of the process, and we provide a starting point for those willing to spend the time to optimize. In our view, poor documentation of tissue and cell type of origin and the use of nonoptimized protocols is a source of inefficiency in current life science research. Even incremental improvement in this area will increase productivity significantly.
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Perfilación de la Expresión Génica/métodos , Neoplasias Pancreáticas/genética , Colorantes , ADN de Neoplasias/genética , Disección/métodos , Humanos , Neoplasias Pancreáticas/patología , ARN Neoplásico/genéticaRESUMEN
Frozen tissue specimens are the gold standard for molecular analysis. However, snap freezing presents several challenges regarding collection and storage of tissue, and preservation of histological detail. We evaluate an alternative preservation method, ethanol fixation followed by paraffin embedding, by analyzing expression profiles of microdissected cells on Affymetrix oligonucleotide arrays of three matched benign prostatic hyperplasia (BPH) and tumor samples processed with each preservation method. Frozen samples generated an average present call of 26% of the probe sets, compared to 4.5% in ethanol-paraffin samples. Eighty-eight percent of the probe sets called present in the ethanol-paraffin samples were also present in the frozen specimens. Comparing ethanol-paraffin BPH to tumor, 52 probe sets showed a twofold differential expression or higher in at least two cases, 23 of which were also differentially expressed in at least one frozen case. Despite a significant drop in the number of transcripts detectable, the data suggests that the obtainable information in ethanol-fixed samples may be useful for molecular profiling where frozen tissue is not available. However, ethanol fixation and paraffin embedding of tissue specimens is not optimal for high-throughput mRNA expression analysis. Improved methods for transcript profiling of archival samples, and/or tissue processing are still required.
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Etanol/farmacología , Regulación de la Expresión Génica , Próstata/patología , Hiperplasia Prostática/patología , Manejo de Especímenes/métodos , Fijación del Tejido/métodos , Fijadores , Congelación , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Rayos Láser , Masculino , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Parafina/química , Adhesión en Parafina/métodos , ARN/metabolismo , ARN Mensajero/metabolismo , ARN NeoplásicoRESUMEN
Proteomics, the global study of protein expression and characteristics, has recently emerged as a key component in the field of molecular analysis. The dynamic nature of proteins, from ion channels to chaperones, presents a challenge, yet the understanding of these molecules provides a rich source of information. When applying proteomic analysis directly to human tissue samples, additional difficulties arise. The following article presents an overview of the current proteomic tools used in the analysis of tissues, beginning with conventional methods such as western blot analysis and 2D polyacrylamide gel electrophoresis. The most current high-throughput techniques being used today are also reviewed. These include protein arrays, reverse-phase protein lysate arrays, matrix-assisted laser desorption/ionization, surface-enhanced laser desorption/ionization and layered expression scanning. In addition, bioinformatics as well as issues regarding tissue preservation and microdissection to obtain pure cell populations are included. Finally, future directions of the tissue proteomics field are discussed.
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Proteoma/química , Proteómica/tendencias , Animales , Análisis por Micromatrices/métodos , Proteómica/métodos , ARN/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Tissue microdissection is an important method for the study of disease states. However, it is difficult to perform high-throughput molecular analysis with current techniques. We describe here a prototype version of a novel technique (expression microdissection) that allows for the procurement of desired cells via molecular targeting. Expression microdissection (xMD) offers significant advantages over available methods, including an increase in dissection speed of several orders of magnitude. xMD may become a valuable tool for investigators studying cancer or other disease states in patient specimens and animal models.