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2.
Acta Cytol ; 64(1-2): 81-91, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30889574

RESUMO

Fine needle aspiration samples and small biopsies provide a minimally invasive diagnostic modality for mass lesions. When an infectious process is suspected based on initial evaluation, ancillary techniques can assist in making a specific diagnosis. Here we review the cytopathology that should prompt additional testing and review the availability and interpretation of special stains, immunohistochemistry, and in situ hybridization. In addition, this review addresses when special cultures may be necessary and the use of newer molecular techniques for pathogen identification.


Assuntos
Biópsia por Agulha Fina/métodos , Citodiagnóstico/métodos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Bactérias/genética , Bactérias/isolamento & purificação , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Humanos , Técnicas de Diagnóstico Molecular/métodos , Guias de Prática Clínica como Assunto
3.
Acta Cytol ; 64(1-2): 30-39, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30783052

RESUMO

Human papilloma virus (HPV)-related squamous cell carcinoma (SCC) is biologically unique and has a better prognosis than conventional SCC of the head and neck. p16 immunohistochemistry emerged as a valuable surrogate marker for HPV in oropharyngeal SCC. The criteria for a positive p16 result in tissue specimens are well established. However, there is no consensus regarding interpreting p16 staining in cell blocks and other cytology specimens. This review discusses the current evidence on p16 testing in cytology specimens and also highlights other methods for HPV testing, including DNA and RNA in situ hybridization, as well as other molecular HPV tests.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Imuno-Histoquímica/métodos , Infecções por Papillomavirus/metabolismo , Biópsia por Agulha Fina/métodos , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Hibridização In Situ/métodos , Papillomaviridae/genética , Papillomaviridae/fisiologia , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Sensibilidade e Especificidade
4.
Nucleic Acids Res ; 48(3): e17, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31853536

RESUMO

Multiplexed RNA in situ hybridization for the analysis of gene expression patterns plays an important role in investigating development and disease. Here, we present a method for multiplexed RNA-ISH to detect spatial tumor heterogeneity in tissue sections. We made use of a microfluidic chip to deliver ISH-probes locally to regions of a few hundred micrometers over time periods of tens of minutes. This spatial multiplexing method can be combined with ISH-approaches based on signal amplification, with bright field detection and with the commonly used format of formalin-fixed paraffin-embedded tissue sections. By using this method, we analyzed the expression of HER2 with internal positive and negative controls (ActB, dapB) as well as predictive biomarker panels (ER, PgR, HER2) in a spatially multiplexed manner on single mammary carcinoma sections. We further demonstrated the applicability of the technique for subtype differentiation in breast cancer. Local analysis of HER2 revealed medium to high spatial heterogeneity of gene expression (Cohen effect size r = 0.4) in equivocally tested tumor tissues. Thereby, we exemplify the importance of using such a complementary approach for the analysis of spatial heterogeneity, in particular for equivocally tested tumor samples. As the method is compatible with a range of ISH approaches and tissue samples, it has the potential to find broad applicability in the context of molecular analysis of human diseases.


Assuntos
Hibridização In Situ/métodos , Técnicas Analíticas Microfluídicas/métodos , RNA Neoplásico/análise , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Neoplasias da Mama/química , Neoplasias da Mama/classificação , Linhagem Celular , Feminino , Humanos , Dispositivos Lab-On-A-Chip , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo
5.
Virchows Arch ; 475(6): 757-762, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31673776

RESUMO

Epstein-Barr virus (EBV) has been associated with about 9% of all gastric carcinomas, but its role in gastric carcinogenesis remains unclear since there is lack of evidence of EBV presence in pre-neoplastic lesions of gastric mucosa. This study intends to determine the prevalence of EBV in gastric dysplasia and superficial neoplasia to clarify whether EBV infection is an early or late event in gastric cancer development. This retrospective study included a total of 242 gastric lesions from 199 consecutive patients who were referred for endoscopic resection. The histological classification of lesions includes 137 low- and high-grade dysplasia and 105 superficial carcinomas. EBV infection was investigated by EBER-ISH. Results showed that EBV was not detected in any epithelial cells of any case with dysplasia or superficial carcinomas, although we observed the presence of a small number of EBV-infected lymphocytes in 2.1% of all lesions. These results showed that EBV is not present in gastric dysplasia neither in superficial carcinomas suggesting that EBV carcinogenesis is a late event in well/moderately differentiated gastric carcinogenesis.


Assuntos
Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/patogenicidade , Neoplasias Gástricas/virologia , Estômago/virologia , Idoso , Carcinoma/patologia , Carcinoma/virologia , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/patologia , Feminino , Mucosa Gástrica/patologia , Mucosa Gástrica/virologia , Humanos , Hibridização In Situ/métodos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Estômago/patologia , Neoplasias Gástricas/patologia
6.
Pediatr Surg Int ; 35(12): 1329-1338, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31570973

RESUMO

PURPOSE: Epigenetic factors are involved in the pathogenesis of congenital diaphragmatic hernia (CDH). Circular RNAs (circRNAs) are epigenetic regulators amenable to biomarker profiling. Here, we aimed to develop a liquid biopsy protocol to detect pathognomonic circRNA changes in biofluids. METHODS: Our protocol is adapted from the existing BaseScope™ in situ hybridization technique. Rat biofluids were fixed in a gelatin-coated 96-well plate with formalin. Probes were designed to target circRNAs with significant fold change in nitrofen-induced CDH. FastRED fluorescence was assessed using a plate reader and confirmed with confocal microscopy. We tested maternal serum and amniotic fluid samples from control and nitrofen-treated rats. RESULTS: We detected circRNAs in rat serum and amniotic fluid from control and CDH (nitrofen-treated) rats using fluorescent readout. CircRNA signal was observed in fixed biofluids as fluorescent punctate foci under confocal laser scanning microscopy. This was confirmed by comparison to BaseScope™ lung tissue sections. Signal was concentration dependent and DNase resistant. CONCLUSION: We successfully adapted BaseScope™ to detect circRNAs in rat biofluids: serum and amniotic fluid. We detected signal from probes targeted to circRNAs that are dysregulated in rat CDH. This work establishes the preliminary feasibility of circRNA detection in prenatal diagnostics.


Assuntos
Hérnias Diafragmáticas Congênitas/metabolismo , Hérnias Diafragmáticas Congênitas/patologia , Hibridização In Situ/métodos , /metabolismo , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Feminino , Hérnias Diafragmáticas Congênitas/diagnóstico , Biópsia Líquida , Gravidez , Ratos , Ratos Sprague-Dawley
7.
Cardiovasc Pathol ; 43: 107142, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31442826

RESUMO

Intimal sarcoma is a rare malignant mesenchymal tumor arising from the intima of the great vessels and the heart, and is associated with poor outcomes. As clinico-radiological findings and pathological features are often non-specific, the diagnosis of intimal sarcoma is challenging. Recently, MDM2 amplification was reported to be a characteristic genetic event in this tumor. In the present study, we examined MDM2 status by immunohistochemistry, and by fluorescence and dual-color in situ hybridization (FISH and DISH) using intimal sarcoma (10 tumors), angiosarcoma (5), pulmonary sarcomatoid carcinoma (p-SC) (14) and chronic pulmonary thrombosis (CPT) (3) to investigate MDM2 amplification for the diagnosis of intimal sarcoma. MDM2 and CDK4 were immunopositive in all 10 intimal sarcoma tumors, and high-level amplification of MDM2 was detected in eight tumors by both FISH and DISH. The other two tumors had polysomy of chromosome 12 and overexpression of p53 protein. Although MDM2 aberrations were observed in three p-SCs (two with amplification and one with polysomy), angiosarcomas and CPTs lacked MDM2 amplification. Furthermore, there was high concordance between FISH and DISH. In conclusion, we found that MDM2 amplification strongly supports the diagnosis of intimal sarcoma, and MDM2 DISH was a concordant method and an acceptable alternative to FISH. As MDM2 amplification and p53 overexpression were mutually exclusive, disruption of the MDM2-p53 pathway may be an essential genetic event for this malignant tumor.


Assuntos
Biomarcadores Tumorais/genética , Amplificação de Genes , Neoplasias Cardíacas/genética , Hibridização In Situ/métodos , Proteínas Proto-Oncogênicas c-mdm2/genética , Sarcoma/genética , Túnica Íntima/enzimologia , Neoplasias Vasculares/genética , Adulto , Idoso , Feminino , Neoplasias Cardíacas/enzimologia , Neoplasias Cardíacas/patologia , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Sarcoma/enzimologia , Sarcoma/patologia , Túnica Íntima/patologia , Neoplasias Vasculares/enzimologia , Neoplasias Vasculares/patologia
8.
Malays J Pathol ; 41(2): 133-138, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31427548

RESUMO

INTRODUCTION: Evaluation of HER2 status in breast cancer using immunohistochemistry (IHC) and in-situ-hybridisation (ISH) study is important to establish prognosis and to select patient for targeted therapy. OBJECTIVE: The study aims to determine the concordance between HER2 protein IHC score and its gene status by dual-colour dual-hapten in-situ-hybridization (DDISH) study. MATERIALS AND METHODS: Retrospective study was performed on 767 referred breast cancer cases over a period of five years. The HER2 IHC score (the initial and repeat test score) and the results of HER2 gene status by DDISH were retrieved from the histopathological reports. The agreement between initial IHC score with repeat test score was measured using Cohen Kappa. Chi square test analyzed the association between HER2 IHC score with its gene status by DDISH. RESULTS: The concordance of HER2 IHC score between the initial and repeat test were 52.7% and 89.4% for IHC score 2+ and 3+ respectively. There was moderate agreement of HER2 IHC score between the initial and repeat test score (Ï° = 0.526, p<0.001). A significant association noted between HER2 IHC score with its gene status by DDISH (p<0.001). Only 56 out of 207 cases (27.1%) with 2+ IHC score showed HER2 gene amplification while the majority of cases with 3+ IHC score were gene-amplified (446 out of 451, 98.9%). CONCLUSION: ISH study should be done in all IHC-equivocal cases (2+) to select patient for targeted therapy. Gene amplification must also be confirmed in IHC-positive cases (3+) to prevent from giving non-effective treatment with possible adverse effects to patient with non-amplified HER2 gene.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Receptor ErbB-2/análise , Adulto , Neoplasias da Mama/genética , Feminino , Humanos , Pessoa de Meia-Idade , Receptor ErbB-2/biossíntese , Reprodutibilidade dos Testes , Estudos Retrospectivos
9.
Semin Diagn Pathol ; 36(5): 336-341, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31227426

RESUMO

In situ hybridization (ISH) has become a common laboratory technique used for the analysis of gene expression and for the localization of specific DNA and RNA molecules in cells. Many different methods of performing ISH have been described. These techniques have evolved into important tools in basic scientific research and in clinical diagnoses. One of the goals of ISH is to localize gene sequences in situ and to visualize the products within cells while preserving cell integrity. This allows for meaningful anatomical and histological interpretation of the localized product(s) within heterogeneous tissues. Because of the possibility of false positive and false negative results that may occur with ISH assays, familiarity with the pathophysiology of the molecules that are analyzed and the cellular processes involved as well as with limitations of the assays can help to avoid erroneous diagnoses with clinical specimens.


Assuntos
Perfilação da Expressão Gênica/métodos , Hibridização In Situ/métodos , Humanos
10.
Curr Protoc Mol Biol ; 127(1): e93, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31237425

RESUMO

MicroRNAs (miRNAs) are key regulators of cell and tissue development. However, spatial resolution of miRNA heterogeneity and accumulation patterns in vivo remains uncharted. Next-generation sequencing methods assay miRNA abundance in tissues, yet these analyses do not provide spatial resolution. A method to assay miRNA expression at single-cell resolution in vivo should clarify the cell-autonomous functions of miRNAs, their roles in influencing the cellular microenvironment, and their perdurance and turnover rate. We present an in situ hybridization protocol to map miRNA subcellular expression in single cells in vivo in four days. Using this protocol, we mapped distinct miRNAs that accumulate in the cytoplasm of one sibling oocyte but not another, dependent on the oocyte developmental stage. Thus, this method provides spatial and temporal resolution of the heterogeneity in expression of miRNAs during Caenorhabditis elegans oogenesis. This protocol can generally be adapted to any tissue amenable to dissection and fixation. © 2019 by John Wiley & Sons, Inc.


Assuntos
Caenorhabditis elegans/genética , MicroRNAs/genética , Oócitos/metabolismo , Oogênese/genética , Análise de Célula Única/métodos , Animais , Hibridização In Situ/métodos , Oócitos/citologia
11.
RNA ; 25(9): 1211-1217, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31209064

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD) is among the most common forms of muscular dystrophy. FSHD is caused by aberrant expression of the toxic DUX4 gene in muscle. Detecting endogenous DUX4 in patient tissue using conventional methods can be challenging, due to the low level of DUX4 expression. Therefore, developing simple and trustworthy DUX4 detection methods is an important need in the FSHD field. Here, we describe such a method, which uses the RNAscope assay, an RNA in situ hybridization (ISH) technology. We show that a custom-designed RNAscope assay can detect overexpressed DUX4 mRNA in transfected HEK293 cells and endogenous DUX4 mRNA in FSHD patient-derived myotubes. The RNAscope assay was highly sensitive for tracking reductions in DUX4 mRNA following treatment with our therapeutic mi405 microRNA, suggesting that RNAscope-based DUX4 expression assays could be developed as a prospective outcome measure in therapy trials. This study could set the stage for optimizing and developing a new, rapid RNA ISH-based molecular diagnostic assay for future clinical use in the FSHD field.


Assuntos
Proteínas de Homeodomínio/genética , Hibridização In Situ/métodos , RNA/genética , Linhagem Celular , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular Facioescapuloumeral/genética , Patologia Molecular/métodos , RNA Mensageiro/genética
12.
J Vet Intern Med ; 33(4): 1660-1668, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31169944

RESUMO

BACKGROUND: A recent genome-wide association study in German Shepherd dogs (GSDs) with chronic enteropathy (CE) has identified polymorphisms in the Th2 cytokine genes. HYPOTHESIS/OBJECTIVE: To determine if the expression of the Th2 cytokines, interleukin-13 (IL-13) and interleukin-33 (IL-33), is altered in the duodenal mucosa of GSDs with CE compared to non-GSDs with CE and healthy dogs. ANIMALS: Twenty client-owned dogs diagnosed with CE (10 GSDs and 10 non-GSDs) at the Bristol Veterinary School and 8 healthy Beagle dogs from the Iowa State University Service Colony. METHODS: Retrospective study using archived paraffin-embedded duodenal biopsy samples. A novel RNA in situ hybridization technology (RNAscope) was used to hybridize IL-13 and IL-33 mRNA probes onto at least 10 sections from duodenal biopsy samples for each dog. RNAscope signals were visualized using a microscope and semi-quantitative assessment was performed by a single operator. RESULTS: Based on duodenal villus, subvillus, epithelial, and lamina propria average expression scores, GSDs with CE had significantly lower IL-13 and IL-33 mRNA expression compared to non-GSDs with CE (IL-13, P < .04; IL-33, P < .02) and healthy Beagle dogs (IL-13, P < .02; IL-33, P < .004). CONCLUSIONS AND CLINICAL IMPORTANCE: Similar to human patients with ulcerative colitis, a subtype of human inflammatory bowel disease, these data indicate that Th2 cytokines may be involved in the pathogenesis of CE in GSDs.


Assuntos
Doenças do Cão/metabolismo , Interleucina-13/genética , Interleucina-33/genética , Enteropatias/veterinária , Mucosa Intestinal/metabolismo , Animais , Doenças do Cão/genética , Cães , Duodeno/metabolismo , Feminino , Hibridização In Situ/métodos , Hibridização In Situ/veterinária , Interleucina-13/metabolismo , Interleucina-33/metabolismo , Masculino , RNA Mensageiro/metabolismo , Estudos Retrospectivos
13.
Molecules ; 24(11)2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31159269

RESUMO

We describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, Staphylococcus aureus (S. aureus) was selected as a model for our study to show the ability of this probe to bind to intracellular 16S ribosomal rRNA. A highly purified Europium chelate conjugated oligonucleotide probe complementary to an rRNA sequence-specific S. aureus was prepared and found to be soluble and stable in aqueous solution. The probe was able to bind specifically to S. aureus via in situ hybridisation to differentiate S. aureus from a closely related but less pathogenic Staphylococcus species (S. epidermidis). A time-gated luminescent (TGL) microscope system was used to generate the high signal-to-noise ratio (SNR) images of the S. aureus. After excitation (365 nm, Chelate λmax = 335 nm), the long-lived (Eu3+) luminescent emission from the probe was detected without interference from natural background autofluorescence typically seen in biological samples. The luminescent images were found to have 6 times higher SNR or sensitivity compared to the fluorescent images using conventional fluorophore Alexa Fluor 488. The TEGylated Europium chelate -oligo probe stained S. aureus with mean signal intensity 3.5 times higher than the threshold level of signal from S. epidermidis (with SNR 8 times higher). A positive control probe (EUB338-BHHTEGST-Eu3+) has mean signal intensity for S. aureus and S. epidermidis equally 3.2 times higher than the threshold of signal for a negative NON-EUB338 control probe. The direct conjugation of a single Europium chelate to DNA provides simplicity and improvement over existing bovine serum albumin (BSA)/streptavidin/biotinylated DNA platforms for multi-attachment of Europium chelate per DNA and more importantly makes it feasible for hybridisation to intracellular RNA targets. This probe has great potential for highly sensitive homogeneous in situ hybridisation detection of the vast range of intracellular DNA targets.


Assuntos
Hibridização In Situ , Luminescência , Medições Luminescentes , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Cromatografia Líquida de Alta Pressão , Humanos , Hibridização In Situ/métodos , Medições Luminescentes/métodos , RNA Ribossômico 16S
14.
Zoolog Sci ; 36(1): 58-67, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31116539

RESUMO

Here, we demonstrated an antagonistic effect of short neuropeptide F (sNPF) in modulating feeding motivation in the silkworm Bombyx mori; sNPF reduced the feeding-delaying effects caused by administration of an inhibitory peptide, allatotropin (AT). In situ hybridization and MALDI-TOF MS analysis revealed the presence of three subtypes of sNPFs (sNPF-1, -2, and -3) in the midgut enteroendocrine cells. Ca2+-imaging analyses revealed that three subtypes of sNPF receptors (sNPFRs) (BNGR-A7, -A10, and -A11) showed different affinities with the three subtypes of sNPFs. In addition, sNPF activated its signaling via ERK phosphorylation in the midgut, while mixture of sNPF and AT reduced the phosphorylation level, agreeing with the results of behavioral assay. Together, our current findings suggest that intestinal sNPF positively modulates the feeding motivation by reducing the inhibitory effects by AT within the midgut.


Assuntos
Comportamento Alimentar/efeitos dos fármacos , Trato Gastrointestinal/efeitos dos fármacos , Hormônios de Inseto/farmacologia , Neuropeptídeos/farmacologia , Animais , Bombyx , Hibridização In Situ/métodos , Larva , Sistema de Sinalização das MAP Quinases , Fosforilação , Receptores de Neuropeptídeos/fisiologia , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
15.
J Clin Pathol ; 72(9): 603-608, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31129615

RESUMO

AIMS: Human epidermal growth factor receptor 2 (HER2)-targeted agents are effective against HER2-positive breast cancers. However, their lack of survival benefit in HER2-negative patients as well as their toxic effects and high cost highlight the need for accurate assessment of HER2 status. Our aim was to evaluate the clinical utility of a reagent-saving in situ hybridisation (Saving ISH) that facilitates hybridisation and saves HER2/chromosome enumeration probe by taking advantage of the non-contact mixing effect of an alternating current (AC) electric field. METHODS: With a new device, we apply a high-voltage, low-frequency AC electric field to the tissue sections, which mixes the probe within microdroplets as the voltage is switched on and off. Specimens (n=113) from patients with breast cancers identified immunohistochemically as HER2 0/1(+), (2+) or (3+) were used. The specimens were all tested using conventional dual ISH (DISH), DISH with an automated slide stainer (ASS) and Saving ISH (1:1-1:3 dilution). RESULTS: The Saving ISH with 1:2 probe dilution produced stable results with less non-specific staining while using smaller amounts of probe. The accuracy of HER2 status with Saving ISH was equal to standard. We found 96.4% agreement between DISH using ASS and Saving ISH (kappa coefficient=0.912). CONCLUSIONS: These results suggest reagent-saving HER2 ISH could be used as a clinical tool for accurate and stable HER2 assessment, even when reagent concentrations vary.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Eletricidade , Amplificação de Genes , Hibridização In Situ/métodos , Receptor ErbB-2/genética , Biomarcadores Tumorais/análise , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Desenho de Equipamento , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ/instrumentação , Valor Preditivo dos Testes , Receptor ErbB-2/análise , Reprodutibilidade dos Testes
16.
Methods Mol Biol ; 1933: 99-130, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945181

RESUMO

(m)RNA spatiotemporal pattern of distribution is of key importance to decipher gene function. In this post-genomic era, numerous transcriptomic studies are made publicly available, sometimes reaching a tissular resolution and even more rarely the cellular level. This "one tissue-numerous genes" information can be completed by the reverse "one gene-numerous tissues" picture through traditional RNA in situ hybridization (ISH). Here, we present a method including (1) principles of transcriptomic data mining to be performed prior and following ISH and (2) a detailed step-by-step medium-throughput ISH protocol performed on serial sections from tissue microarrays. In a recent work, we implemented this method for 39 selected genes studied by medium-throughput ISH complementing an existing tissue-specific transcriptomic dataset focused on the model plant Arabidopsis seed development kinetics (Francoz et al., Scientific Reports 6:24644, 2016). This full integration of ISH and transcriptomics demonstrated the complementarity of both techniques in terms of tissue/cell specificity, signal sensitivity, gene specificity, and spatiotemporal resolution.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Hibridização In Situ/métodos , Inclusão em Parafina/métodos , Sondas RNA/química , RNA de Plantas/genética , Análise Serial de Tecidos/métodos , Especificidade de Órgãos
17.
Methods Cell Biol ; 151: 177-196, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948007

RESUMO

A critical process in embryonic development is the activation and spatial localization of mRNAs to specific cells and territories of the embryo. Revealing the spatial distribution of mRNAs and how it changes during development is a vital piece of information that aids in understanding the signaling and regulatory genes driving specific gene regulatory networks. In the laboratory, a cost-efficient, reliable method to determine the spatial distribution of mRNAs in embryos is in situ hybridization. This sensitive and straightforward method employs exogenous antisense RNA probes to find specific and complementary sequences in fixed embryos. Antigenic moieties conjugated to the ribonucleotides incorporated in the probe cross-react with antibodies, and numerous staining methods can be subsequently employed to reveal the spatial distribution of the targeted mRNA. The quality of the data produced by this method is equivalent to the experience of the researcher, and thus a thorough understanding of the numerous steps comprising this method is important for obtaining high quality data. Here we compile and summarize several protocols that have been employed chiefly on five sea urchin species in numerous laboratories around the world. Whereas the protocols can vary for the different species, the overarching steps are similar and can be readily mastered. When properly and carefully undertaken, in situ hybridization is a powerful tool providing unambiguous data for which there currently is no comparable substitute and will continue to be an important method in the era of big data and beyond.


Assuntos
Desenvolvimento Embrionário/genética , Redes Reguladoras de Genes/genética , Hibridização In Situ/métodos , Ouriços-do-Mar/genética , Animais , Embrião não Mamífero/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Larva/genética , Larva/crescimento & desenvolvimento , RNA Mensageiro/genética , Ouriços-do-Mar/crescimento & desenvolvimento
18.
Virchows Arch ; 475(3): 303-311, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30953146

RESUMO

To evaluate the effect of the 2018 ASCO/CAP guideline in the identification of HER2-positive breast carcinomas (BC) in reflex in situ hybridization (ISH) test. A total of 592 primary invasive BC cases from before and after the publication of the updated ASCO/CAP guideline were evaluated for HER2 amplification by silver ISH according to the 2013 and 2018 guidelines. Cases were mostly (95%) HER2 equivocal by immunohistochemistry (IHC), not centrally reviewed. Other reasons for referring cases were IHC confirmation, IHC discordancy (either between needle-core-biopsy (NCB) and surgical excision specimen (SES) or between different laboratories) and IHC result unexpected for histopathologic features. Cases evaluated with the 2013 guideline (1st cohort) were 14.6% HER2-positive, decreasing significantly after the reclassification with the 2018 guideline due to the exclusion of group 2 cases without HER2 protein overexpression. Cases studied after the implementation of the 2018 guideline (2nd cohort) were 8.7% HER2-positive, a frequency that was not significantly different from the reclassification of the 1st cohort with the 2018 guideline. All cases referred for IHC confirmation had the expected ISH result. Cases with IHC discordancy between NCB and SES were ISH concordant. Only one out of 14 cases with an IHC score 3+ and classified as histological grade 1 or with a Ki67 below 10% was classified as ISH HER2-positive. The 2018 ASCO/CAP guideline resulted in a decrease of HER2-positive cases in reflex ISH test, selecting less patients for anti-HER2-targeted therapy.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Hibridização in Situ Fluorescente/métodos , Biópsia com Agulha de Grande Calibre/métodos , Feminino , Humanos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Patologia Clínica/normas , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo
20.
J Vis Exp ; (145)2019 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-30882790

RESUMO

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.


Assuntos
Characidae/embriologia , Characidae/genética , Embrião não Mamífero/metabolismo , Hibridização In Situ/métodos , Animais , Evolução Biológica , Cavernas , Locos de Características Quantitativas
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