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1.
J Biomol Tech ; 28(1): 31-39, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28337070

RESUMO

The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Resource Facilities Metagenomics Research Group (ABRF MGRG) that focuses on whole genome shotgun sequencing of extreme and unique environments using a wide variety of biomolecular techniques. The goals are multifaceted, including development and refinement of new techniques for the following: 1) the detection and characterization of novel microbes, 2) the evaluation of nucleic acid techniques for extremophilic samples, and 3) the identification and implementation of the appropriate bioinformatics pipelines. Here, we highlight the different ongoing projects that we have been working on, as well as details on the various methods we use to characterize the microbiome and metagenome of these complex samples. In particular, we present data of a novel multienzyme extraction protocol that we developed, called Polyzyme or MetaPolyZyme. Presently, the XMP is characterizing sample sites around the world with the intent of discovering new species, genes, and gene clusters. Once a project site is complete, the resulting data will be publically available. Sites include Lake Hillier in Western Australia, the "Door to Hell" crater in Turkmenistan, deep ocean brine lakes of the Gulf of Mexico, deep ocean sediments from Greenland, permafrost tunnels in Alaska, ancient microbial biofilms from Antarctica, Blue Lagoon Iceland, Ethiopian toxic hot springs, and the acidic hypersaline ponds in Western Australia.


Assuntos
Microbiologia Ambiental , Microbiota/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Ambientes Extremos , Metagenoma , Tipagem Molecular/normas , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Padrões de Referência , Análise de Sequência de DNA/normas
2.
J Biomol Tech ; 27(2): 75-83, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26977138

RESUMO

The ability to profile expression levels of a large number of mRNAs and microRNAs (miRNAs) within the same sample, using a single assay method, would facilitate investigations of miRNA effects on mRNA abundance and streamline biomarker screening across multiple RNA classes. A protocol is described for reverse transcription of long RNA and miRNA targets, followed by preassay amplification of the pooled cDNAs and quantitative PCR (qPCR) detection for a mixed panel of candidate RNA biomarkers. The method provides flexibility for designing custom target panels, is robust over a range of input RNA amounts, and demonstrated a high assay success rate.


Assuntos
Perfilação da Expressão Gênica/métodos , MicroRNAs/genética , RNA Mensageiro/genética , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Perfilação da Expressão Gênica/instrumentação , Humanos , MicroRNAs/sangue , Técnicas Analíticas Microfluídicas , RNA Mensageiro/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
PLoS One ; 10(2): e0117471, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25710170

RESUMO

HSV is a large double stranded DNA virus, capable of causing a variety of diseases from the common cold sore to devastating encephalitis. Although DNA within the HSV virion does not contain any histone protein, within 1 h of infecting a cell and entering its nucleus the viral genome acquires some histone protein (nucleosomes). During lytic infection, partial micrococcal nuclease (MNase) digestion does not give the classic ladder band pattern, seen on digestion of cell DNA or latent viral DNA. However, complete digestion does give a mono-nucleosome band, strongly suggesting that there are some nucleosomes present on the viral genome during the lytic infection, but that they are not evenly positioned, with a 200 bp repeat pattern, like cell DNA. Where then are the nucleosomes positioned? Here we perform HSV-1 genome wide nucleosome mapping, at a time when viral replication is in full swing (6 hr PI), using a microarray consisting of 50mer oligonucleotides, covering the whole viral genome (152 kb). Arrays were probed with MNase-protected fragments of DNA from infected cells. Cells were not treated with crosslinking agents, thus we are only mapping tightly bound nucleosomes. The data show that nucleosome deposition is not random. The distribution of signal on the arrays suggest that nucleosomes are located at preferred positions on the genome, and that there are some positions that are not occupied (nucleosome free regions -NFR or Nucleosome depleted regions -NDR), or occupied at frequency below our limit of detection in the population of genomes. Occupancy of only a fraction of the possible sites may explain the lack of a typical MNase partial digestion band ladder pattern for HSV DNA during lytic infection. On average, DNA encoding Immediate Early (IE), Early (E) and Late (L) genes appear to have a similar density of nucleosomes.


Assuntos
Genoma Viral , Herpesvirus Humano 1/genética , Nucleossomos/metabolismo , Carbocianinas/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Análise por Conglomerados , Hibridização Genômica Comparativa , Sondas de DNA/metabolismo , DNA Viral/metabolismo , Genes Precoces , Herpesvirus Humano 1/fisiologia , Humanos , Nuclease do Micrococo/metabolismo , Nucleossomos/química , Replicação Viral/genética
5.
Genet Epidemiol ; 39(3): 217-26, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25599974

RESUMO

Preterm birth is the leading cause of infant morbidity and mortality. Despite extensive research, the genetic contributions to spontaneous preterm birth (SPTB) are not well understood. Term controls were matched with cases by race/ethnicity, maternal age, and parity prior to recruitment. Genotyping was performed using Affymetrix SNP Array 6.0 assays. Statistical analyses utilized PLINK to compare allele occurrence rates between case and control groups, and incorporated quality control and multiple-testing adjustments. We analyzed DNA samples from mother-infant pairs from early SPTB cases (20(0/7)-33(6/7) weeks, 959 women and 979 neonates) and term delivery controls (39(0/7)-41(6/7) weeks, 960 women and 985 neonates). For validation purposes, we included an independent validation cohort consisting of early SPTB cases (293 mothers and 243 infants) and term controls (200 mothers and 149 infants). Clustering analysis revealed no population stratification. Multiple maternal SNPs were identified with association P-values between 10×10(-5) and 10×10(-6). The most significant maternal SNP was rs17053026 on chromosome 3 with an odds ratio (OR) 0.44 with a P-value of 1.0×10(-6). Two neonatal SNPs reached the genome-wide significance threshold, including rs17527054 on chromosome 6p22 with a P-value of 2.7×10(-12) and rs3777722 on chromosome 6q27 with a P-value of 1.4×10(-10). However, we could not replicate these findings after adjusting for multiple comparisons in a validation cohort. This is the first report of a genome-wide case-control study to identify single nucleotide polymorphisms (SNPs) that correlate with SPTB.


Assuntos
Biomarcadores/análise , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único/genética , Nascimento Prematuro/genética , Adulto , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Recém-Nascido , Idade Materna , Paridade , Gravidez , Estudos de Validação como Assunto , Adulto Jovem
6.
MBio ; 5(5): e01714-14, 2014 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-25227467

RESUMO

UNLABELLED: Screening for thousands of viruses and other pathogenic microorganisms, including bacteria, fungi, and parasites, in human tumor tissues will provide a better understanding of the contributory role of the microbiome in the predisposition for, causes of, and therapeutic responses to the associated cancer. Metagenomic assays designed to perform these tasks will have to include rapid and economical processing of large numbers of samples, supported by straightforward data analysis pipeline and flexible sample preparation options for multiple input tissue types from individual patients, mammals, or environmental samples. To meet these requirements, the PathoChip platform was developed by targeting viral, prokaryotic, and eukaryotic genomes with multiple DNA probes in a microarray format that can be combined with a variety of upstream sample preparation protocols and downstream data analysis. PathoChip screening of DNA plus RNA from formalin-fixed, paraffin-embedded tumor tissues demonstrated the utility of this platform, and the detection of oncogenic viruses was validated using independent PCR and deep sequencing methods. These studies demonstrate the use of the PathoChip technology combined with PCR and deep sequencing as a valuable strategy for detecting the presence of pathogens in human cancers and other diseases. IMPORTANCE: This work describes the design and testing of a PathoChip array containing probes with the ability to detect all known publicly available virus sequences as well as hundreds of pathogenic bacteria, fungi, parasites, and helminths. PathoChip provides wide coverage of microbial pathogens in an economical format. PathoChip screening of DNA plus RNA from formalin-fixed, paraffin-embedded tumor tissues demonstrated the utility of this platform, and the detection of oncogenic viruses was validated using independent PCR and sequencing methods. These studies demonstrate that the PathoChip technology is a valuable strategy for detecting the presence of pathogens in human cancers and other diseases.


Assuntos
Bactérias/isolamento & purificação , Fungos/isolamento & purificação , Metagenômica/métodos , Neoplasias/microbiologia , Vírus/isolamento & purificação , Animais , Bactérias/genética , Hibridização Genômica Comparativa , Feminino , Formaldeído/química , Fungos/genética , Humanos , Masculino , Análise em Microsséries , Projetos Piloto , Vírus/genética
7.
Nat Biotechnol ; 32(9): 915-925, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25150835

RESUMO

High-throughput RNA sequencing (RNA-seq) greatly expands the potential for genomics discoveries, but the wide variety of platforms, protocols and performance capabilitites has created the need for comprehensive reference data. Here we describe the Association of Biomolecular Resource Facilities next-generation sequencing (ABRF-NGS) study on RNA-seq. We carried out replicate experiments across 15 laboratory sites using reference RNA standards to test four protocols (poly-A-selected, ribo-depleted, size-selected and degraded) on five sequencing platforms (Illumina HiSeq, Life Technologies PGM and Proton, Pacific Biosciences RS and Roche 454). The results show high intraplatform (Spearman rank R > 0.86) and inter-platform (R > 0.83) concordance for expression measures across the deep-count platforms, but highly variable efficiency and cost for splice junction and variant detection between all platforms. For intact RNA, gene expression profiles from rRNA-depletion and poly-A enrichment are similar. In addition, rRNA depletion enables effective analysis of degraded RNA samples. This study provides a broad foundation for cross-platform standardization, evaluation and improvement of RNA-seq.


Assuntos
Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transcriptoma
8.
J Biomol Tech ; 24(4): 198-217, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24294113

RESUMO

A microarray (LungCaGxE), based on Illumina BeadChip technology, was developed for high-resolution genotyping of genes that are candidates for involvement in environmentally driven aspects of lung cancer oncogenesis and/or tumor growth. The iterative array design process illustrates techniques for managing large panels of candidate genes and optimizing marker selection, aided by a new bioinformatics pipeline component, Tagger Batch Assistant. The LungCaGxE platform targets 298 genes and the proximal genetic regions in which they are located, using ≈ 13,000 DNA single nucleotide polymorphisms (SNPs), which include haplotype linkage markers with a minimum allele frequency of 1% and additional specifically targeted SNPs, for which published reports have indicated functional consequences or associations with lung cancer or other smoking-related diseases. The overall assay conversion rate was 98.9%; 99.0% of markers with a minimum Illumina design score of 0.6 successfully generated allele calls using genomic DNA from a study population of 1873 lung-cancer patients and controls.


Assuntos
Interação Gene-Ambiente , Neoplasias Pulmonares/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polimorfismo de Nucleotídeo Único , Estudos de Casos e Controles , Técnicas de Genotipagem , Humanos
10.
Am J Obstet Gynecol ; 202(4): 386.e1-6, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20350647

RESUMO

OBJECTIVE: We determined whether an environmental exposure to bacterial vaginosis (BV) modified genetic susceptibilities for spontaneous preterm delivery within genes that regulate the inflammatory response. STUDY DESIGN: Maternal DNA samples and vaginal smears for Gram staining were collected from 743 women (68 preterm births). We used a 1536-single nucleotide polymorphism (SNP) custom chip to study associations between genotype distributions and preterm birth. RESULTS: For 8 SNPs in 3 genes (protein kinase C alpha, fms-like tyrosine kinase 1, and interleukin 6), the odds ratios for preterm birth ranged from 1.9-4.0 among women with susceptible genotypes who were BV positive. The odds ratios for preterm birth were 2.0-5.0 times greater among women who were BV positive than among women who were BV negative. The significance of these differences was demonstrated by logistic regression analyses for genotype/BV interaction. CONCLUSION: These results demonstrate that the risk of preterm delivery that is associated with tag SNPs in genes that regulate the inflammatory response is modified by an environmental exposure such as bacterial vaginosis.


Assuntos
Variação Genética , Interleucina-6/genética , Nascimento Prematuro , Proteína Quinase C-alfa/genética , Vaginose Bacteriana , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Adolescente , Adulto , Meio Ambiente , Feminino , Frequência do Gene , Predisposição Genética para Doença/epidemiologia , Genótipo , Humanos , Recém-Nascido , Modelos Logísticos , Razão de Chances , Polimorfismo de Nucleotídeo Único , Gravidez , Nascimento Prematuro/epidemiologia , Nascimento Prematuro/genética , Nascimento Prematuro/imunologia , Fatores de Risco , Esfregaço Vaginal , Vaginose Bacteriana/epidemiologia , Vaginose Bacteriana/genética , Vaginose Bacteriana/imunologia , Adulto Jovem
11.
J Clin Invest ; 119(12): 3556-72, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19959874

RESUMO

Natural SIV infection of sooty mangabeys (SMs) is nonprogressive despite chronic virus replication. Strikingly, it is characterized by low levels of immune activation, while pathogenic SIV infection of rhesus macaques (RMs) is associated with chronic immune activation. To elucidate the mechanisms underlying this intriguing phenotype, we used high-density oligonucleotide microarrays to longitudinally assess host gene expression in SIV-infected SMs and RMs. We found that acute SIV infection of SMs was consistently associated with a robust innate immune response, including widespread upregulation of IFN-stimulated genes (ISGs) in blood and lymph nodes. While SMs exhibited a rapid resolution of ISG expression and immune activation, both responses were observed chronically in RMs. Systems biology analysis indicated that expression of the lymphocyte inhibitory receptor LAG3, a marker of T cell exhaustion, correlated with immune activation in SIV-infected RMs but not SMs. Our findings suggest that active immune regulatory mechanisms, rather than intrinsically attenuated innate immune responses, underlie the low levels of immune activation characteristic of SMs chronically infected with SIV.


Assuntos
Cercocebus atys/genética , Cercocebus atys/imunologia , Imunidade Inata/genética , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/patogenicidade , Imunidade Adaptativa/genética , Animais , Antígenos CD/genética , Linfócitos T CD4-Positivos/imunologia , Cercocebus atys/virologia , Estudo de Associação Genômica Ampla , Interferons/genética , Macaca mulatta , Análise de Sequência com Séries de Oligonucleotídeos , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Especificidade da Espécie , Regulação para Cima
12.
J Infect Dis ; 197(8): 1171-84, 2008 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-18462164

RESUMO

Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a multisystem disease, the pathogenesis of which remains undetermined. We set out to determine the precise abnormalities of gene expression in the blood of patients with CFS/ME. We analyzed gene expression in peripheral blood from 25 patients with CFS/ME diagnosed according to the Centers for Disease Control and Prevention diagnostic criteria and 50 healthy blood donors, using a microarray with a cutoff fold difference of expression of >or=2.5. Genes showing differential expression were further analyzed in 55 patients with CFS/ME and 75 healthy blood donors, using quantitative polymerase chain reaction. Differential expression was confirmed for 88 genes; 85 were upregulated, and 3 were downregulated. Highly represented functions were hematological disease and function, immunological disease and function, cancer, cell death, immune response, and infection. Clustering of quantitative polymerase chain reaction data from patients with CFS/ME revealed 7 subtypes with distinct differences in Medical Outcomes Survey Short Form-36 scores, clinical phenotypes, and severity.


Assuntos
Síndrome de Fadiga Crônica/genética , Adulto , Análise por Conglomerados , Síndrome de Fadiga Crônica/sangue , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fatores de Transcrição/genética
13.
Endocr Pathol ; 18(3): 163-73, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18058265

RESUMO

microRNAs (miRNAs) are approximately 22 nt RNAs that negatively regulate target gene expression. Their dysregulation has been implicated in the pathogenesis of a number of human cancers, including papillary thyroid carcinoma (PTC). Whereas previous studies using microarray technologies have largely relied on the ability to procure fresh tissue at the time of surgery to characterize miRNA signatures in PTC, we exploited the ability to procure sufficient miRNA from formalin-fixed paraffin-embedded (FFPE) tissue to describe a series of miRNAs whose expression is dysregulated in PTC compared to benign proliferative multinodular goiter (MNG). We identified 13 miRNAs upregulated and 26 miRNAs downregulated in PTC versus MNG. These include miRNA-21, miRNA-31, miRNA-221, and miRNA-222. Their dysregulation was further validated by real time RT-PCR analysis in an independent set of FFPE tissues. Many of these have previously been described in fresh tissue studies as altered in PTC, confirming the utility of this approach. These results further highlight the applicability of miRNA expression patterns as potential markers of human cancer, and our results suggest that FFPE tissues are suitable resources for such miRNA expression analyses. The ability to utilize FFPE tissue in the molecular characterization of human malignancy will unlock a rich resource for future cancer studies.


Assuntos
Carcinoma Papilar/genética , Regulação Neoplásica da Expressão Gênica , Bócio Nodular/genética , MicroRNAs/genética , Inclusão em Parafina , Neoplasias da Glândula Tireoide/genética , Adulto , Idoso , Análise por Conglomerados , Feminino , Formaldeído/farmacologia , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes
14.
PLoS Genet ; 3(8): e143, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17722985

RESUMO

Genomic aberrations recurrent in a particular cancer type can be important prognostic markers for tumor progression. Typically in early tumorigenesis, cells incur a breakdown of the DNA replication machinery that results in an accumulation of genomic aberrations in the form of duplications, deletions, translocations, and other genomic alterations. Microarray methods allow for finer mapping of these aberrations than has previously been possible; however, data processing and analysis methods have not taken full advantage of this higher resolution. Attention has primarily been given to analysis on the single sample level, where multiple adjacent probes are necessarily used as replicates for the local region containing their target sequences. However, regions of concordant aberration can be short enough to be detected by only one, or very few, array elements. We describe a method called Multiple Sample Analysis for assessing the significance of concordant genomic aberrations across multiple experiments that does not require a-priori definition of aberration calls for each sample. If there are multiple samples, representing a class, then by exploiting the replication across samples our method can detect concordant aberrations at much higher resolution than can be derived from current single sample approaches. Additionally, this method provides a meaningful approach to addressing population-based questions such as determining important regions for a cancer subtype of interest or determining regions of copy number variation in a population. Multiple Sample Analysis also provides single sample aberration calls in the locations of significant concordance, producing high resolution calls per sample, in concordant regions. The approach is demonstrated on a dataset representing a challenging but important resource: breast tumors that have been formalin-fixed, paraffin-embedded, archived, and subsequently UV-laser capture microdissected and hybridized to two-channel BAC arrays using an amplification protocol. We demonstrate the accurate detection on simulated data, and on real datasets involving known regions of aberration within subtypes of breast cancer at a resolution consistent with that of the array. Similarly, we apply our method to previously published datasets, including a 250K SNP array, and verify known results as well as detect novel regions of concordant aberration. The algorithm has been fully implemented and tested and is freely available as a Java application at http://www.cbil.upenn.edu/MSA.


Assuntos
Aberrações Cromossômicas , Interpretação Estatística de Dados , Processamento Eletrônico de Dados/métodos , Genoma Humano , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Algoritmos , Mapeamento Cromossômico , Cromossomos Humanos Par 17 , Análise por Conglomerados , Simulação por Computador , Perfilação da Expressão Gênica , Frequência do Gene , Genes erbB-2 , Ligação Genética , Humanos , Modelos Teóricos
15.
Proc Natl Acad Sci U S A ; 103(20): 7801-6, 2006 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-16682618

RESUMO

To investigate multitissue engraftment of human primitive hematopoietic cells and their differentiation in goats, human CD34+ Lin- cord blood cells transduced with a GFP vector were transplanted into fetal goats at 45-55 days of gestation. GFP+ cells were detected in hematopoietic and nonhematopoietic organs including blood, bone marrow, spleen, liver, kidney, muscle, lung, and heart of the recipient goats (1.2-36% of all cells examined). We identified human beta2 microglobulin-positive cells in multiple tissues. GFP+ cells sorted from the perfused liver of a transplant goat showed human insulin-like growth factor 1 gene sequences, indicating that the engrafted GFP+ cells were of human origin. A substantial fraction of cells engrafted in goat livers expressed the human hepatocyte-specific antigen, proliferating cell nuclear antigen, albumin, hepatocyte nuclear factor, and GFP. DNA content analysis showed no evidence for cellular fusion. Long-term engraftment of GFP+ cells could be detected in the blood of goats for up to 2 yr. Microarray analysis indicated that human genes from a variety of functional categories were expressed in chimeric livers and blood. The human/goat xenotransplant model provides a unique system to study the kinetics of hematopoietic stem cell engraftment, gene expression, and possible stem cell plasticity under noninjured conditions.


Assuntos
Antígenos CD34/metabolismo , Diferenciação Celular/fisiologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Perfilação da Expressão Gênica , Cabras , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Transplante Heterólogo , Animais , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fígado/fisiologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , RNA/sangue , Distribuição Tecidual , Quimeras de Transplante
16.
RNA ; 12(2): 187-91, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16373485

RESUMO

microRNAs (miRNAs) are small (approximately 22 nucleotide) regulatory RNAs which play fundamental roles in many biological processes. Recent studies have shown that the expression of many miRNAs is altered in various human tumors and some miRNAs may function as oncogenes or tumor suppressor genes. However, with the exception of glioblastoma multiforme, the expression of miRNAs in brain tumors is unknown. Furthermore, methods to profile miRNAs from formalin-fixed, paraffin-embedded (FFPE) archival tissues or to study their cellular and subcellular localization in FFPE tissues have been lacking. Here we report the coordinated miRNA expression analysis from the tissue level to the subcellular level, using the RAKE (RNA-primed, array-based, Klenow Enzyme) miRNA microarray platform in conjunction with Locked Nucleic Acid (LNA)-based in situ hybridization (LNA-ISH) on archival FFPE human brains and oligodendroglial tumors. The ability to profile miRNAs from archival tissues at the tissue level, by RAKE microarrays, and at the cellular level by LNA-ISH, will accelerate studies of miRNAs in human diseases.


Assuntos
Neoplasias Encefálicas/genética , Encéfalo/fisiologia , Hibridização In Situ/métodos , MicroRNAs/análise , Análise em Microsséries/métodos , Oligodendroglioma/genética , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Cromossomos Humanos Par 1 , Regulação Neoplásica da Expressão Gênica , Humanos , Oligodendroglioma/patologia , Bancos de Tecidos
17.
J Infect Dis ; 192(2): 276-86, 2005 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15962222

RESUMO

This study was undertaken to further examine the role of the host response to parvovirus B19 in the development of symptoms and consequences of viral persistence. Genomic DNA from 42 patients with symptomatic B19 infection was analyzed using the HuSNP assay (Affymetrix), and the results were compared with those from analysis of 53 healthy control individuals. Fifty-seven single-nucleotide polymorphisms were identified that were significantly associated with symptomatic infection. Total RNA from peripheral blood mononuclear cells from 57 B19-seropositive and 13 B19-seronegative donors was analyzed by hybridization to a single-color microarray representing 9522 human genes. Ninety-two genes were shown to be differentially expressed. Differential expression was confirmed in 6 of 38 genes (SKIP, MACF1, SPAG7, FLOT1, c6orf48, and RASSF5) tested using real-time quantitative polymerase chain reaction in a different group of healthy subjects. Genes identified in both studies play a functional role in the cytoskeleton, integrin signaling, and oncosuppression, themes that have been shown to be important in parvovirus infections.


Assuntos
Regulação Viral da Expressão Gênica , Integrinas/fisiologia , Infecções por Parvoviridae/genética , Parvovirus/patogenicidade , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Criança , Pré-Escolar , Citoesqueleto/virologia , Feminino , Predisposição Genética para Doença , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Parvovirus/imunologia , Valores de Referência , Transdução de Sinais/imunologia
18.
Dev Biol ; 272(2): 483-96, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15282163

RESUMO

Studies using low-resolution methods to assess gene expression during preimplantation mouse development indicate that changes in gene expression either precede or occur concomitantly with the major morphological transitions, that is, conversion of the oocyte to totipotent 2-cell blastomeres, compaction, and blastocyst formation. Using microarrays, we characterized global changes in gene expression and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany and likely underlie these transitions. The analysis confirmed previously described processes or events, but more important, EASE revealed new insights. Response to DNA damage and DNA repair genes are overrepresented in the oocyte compared to 1-cell through blastocyst stages and may reflect the oocyte's response to selective pressures to insure genomic integrity; fertilization results in changes in the transcript profile in the 1-cell embryo that are far greater than previously recognized; and genome activation during 2-cell stage may not be as global and promiscuous as previously proposed, but rather far more selective, with genes involved in transcription and RNA processing being preferentially expressed. These results validate this hypothesis-generating approach by identifying genes involved in critical biological processes that can be the subject of a more traditional hypothesis-driven approach.


Assuntos
Blastocisto/fisiologia , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oócitos/fisiologia , Animais , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica/métodos , Processamento de Imagem Assistida por Computador/métodos , Camundongos , Camundongos Endogâmicos , Gravidez , Reprodutibilidade dos Testes , Software , Transcrição Genética
19.
Proc Natl Acad Sci U S A ; 101(6): 1775-80, 2004 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-14736918

RESUMO

The switch from vegetative to reproductive development in plants necessitates a switch in the developmental program of the descendents of the stem cells in the shoot apical meristem. Genetic and molecular investigations have demonstrated that the plant-specific transcription factor and meristem identity regulator LEAFY (LFY) controls this developmental transition by inducing expression of a second transcription factor, APETALA1, and by regulating the expression of additional, as yet unknown, genes. Here we show that the additional LFY targets include the APETALA1-related factor, CAULIFLOWER, as well as three transcription factors and two putative signal transduction pathway components. These genes are up-regulated by LFY even when protein synthesis is inhibited and, hence, appear to be direct targets of LFY. Supporting this conclusion, cis-regulatory regions upstream of these genes are bound by LFY in vivo. The newly identified LFY targets likely initiate the transcriptional changes that are required for the switch from vegetative to reproductive development in Arabidopsis.


Assuntos
Genoma de Planta , Proteínas de Plantas/genética , Proteínas de Homeodomínio/genética , Fenômenos Fisiológicos Vegetais , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Nat Methods ; 1(2): 155-61, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15782179

RESUMO

MicroRNAs (miRNAs) are small regulatory RNAs that serve fundamental biological roles across eukaryotic species. We describe a new method for high-throughput miRNA detection. The technique is termed the RNA-primed, array-based Klenow enzyme (RAKE) assay, because it involves on-slide application of the Klenow fragment of DNA polymerase I to extend unmodified miRNAs hybridized to immobilized DNA probes. We used RAKE to study human cell lines and brain tumors. We show that the RAKE assay is sensitive and specific for miRNAs and is ideally suited for rapid expression profiling of all known miRNAs. RAKE offers unique advantages for specificity over northern blots or other microarray-based expression profiling platforms. Furthermore, we demonstrate that miRNAs can be isolated and profiled from formalin-fixed paraffin-embedded tissue, which opens up new opportunities for analyses of small RNAs from archival human tissue. The RAKE assay is theoretically versatile and may be used for other applications, such as viral gene profiling.


Assuntos
DNA Polimerase I/química , DNA Polimerase I/genética , Perfilação da Expressão Gênica/métodos , MicroRNAs/química , MicroRNAs/genética , Análise em Microsséries/métodos , Linhagem Celular , Desenho de Equipamento , Análise de Falha de Equipamento , Perfilação da Expressão Gênica/instrumentação , Humanos , MicroRNAs/análise , Análise em Microsséries/instrumentação
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