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1.
Breast Cancer Res ; 15(3): R42, 2013 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-23705859

RESUMO

INTRODUCTION: MicroRNAs (miRNAs) are small, non-coding, single-stranded RNAs between 18-22 nucleotides long that regulate gene expression. Expression of miRNAs is altered in tumor compared to normal tissue; there is some evidence that these changes may be reflected in the serum of cancer cases compared to healthy individuals. This has yet to be examined in a prospective study where samples are collected before diagnosis. METHODS: We used Affymetrix arrays to examine serum miRNA expression profiles in 410 participants in the Sister Study, a prospective cohort study of 50,884 women. All women in the cohort had never been diagnosed with breast cancer at the time of enrollment. We compared global miRNA expression patterns in 205 women who subsequently developed breast cancer and 205 women who remained breast cancer-free. In addition within the case group we examined the association of miRNA expression in serum with different tumor characteristics, including hormone status (ER, PR, and HER-2) and lymph node status. RESULTS: Overall, 414 of 1,105 of the human miRNAs on the chip were expressed above background levels in 50 or more women. When the average expression among controls was compared to cases using conditional logistic regression, 21 miRNAs were found to be differentially expressed (P≤.05). Using qRT-PCR on a small, independent sample of 5 cases and 5 controls we verified overexpression of the 3 highest expressing miRNAs among cases, miR-18a, miR-181a, and miR-222; the differences were not statistically significant in this small set. The 21 differentially expressed miRNAs are known to target at least 82 genes; using the gene list for pathway analysis we found enrichment of genes involved in cancer-related processes. In a separate case-case analyses restricted to the 21 miRNAs, we found 7 miRNAs with differential expression for women whose breast tumors differed by HER-2 expression, and 10 miRNAs with differential expression by nodal status. CONCLUSIONS: miRNA levels in serum show a number of small differences between women who later develop cancer versus those who remain cancer-free.


Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias da Mama/genética , MicroRNAs/biossíntese , Receptor ErbB-2/biossíntese , Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/sangue , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/genética
2.
Pharmaceutics ; 14(7)2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35890393

RESUMO

3DNA holds promise as a carrier for drugs that can be intercalated into its core or linked to surface arms. Coupling 3DNA to an antibody targeting intercellular adhesion molecule 1 (ICAM-1) results in high lung-specific biodistributions in vivo. While the role of individual parameters on ICAM-1 targeting has been studied for other nanocarriers, it has never been examined for 3DNA or in a manner capable of revealing the hierarchic interplay among said parameters. In this study, we used 2-layer vs. 4-layer anti-ICAM 3DNA and radiotracing to examine biodistribution in mice. We found that, below saturating conditions and within the ranges tested, the density of targeting antibodies on 3DNA is the most relevant parameter driving lung targeting over liver clearance, compared to the number of antibodies per carrier, total antibody dose, 3DNA dose, 3DNA size, or the administered concentration, which influenced the dose in organs but not the lung specific-over-liver clearance ratio. Data predicts that lung-specific delivery of intercalating (core loaded) drugs can be tuned using this biodistribution pattern, while that of arm-linked (surface loaded) drugs requires a careful parametric balance because increasing anti-ICAM density reduces the number of 3DNA arms available for drug loading.

3.
Bioeng Transl Med ; 6(2): e10208, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34027094

RESUMO

Biodistribution studies are essential in drug carrier design and translation, and radiotracing provides a sensitive quantitation for this purpose. Yet, for biodegradable formulations, small amounts of free-label signal may arise prior to or immediately after injection in animal models, causing potentially confounding biodistribution results. In this study, we refined a method to overcome this obstacle. First, we verified free signal generation in animal samples and then, mimicking it in a controllable setting, we injected mice intravenously with a radiolabeled drug carrier formulation (125I-antibody/3DNA) containing a known amount of free radiolabel (125I), or free 125I alone as a control. Corrected biodistribution data were obtained by separating the free radiolabel from blood and organs postmortem, using trichloroacetic acid precipitation, and subtracting the confounding signal from each tissue measurement. Control free 125I-radiolabel was detected at ≥85% accuracy in blood and tissues, validating the method. It biodistributed very heterogeneously among organs (0.6-39 %ID/g), indicating that any free 125I generated in the body or present in an injected formulation cannot be simply corrected to the free-label fraction in the original preparation, but the free label must be empirically measured in each organ. Application of this method to the biodistribution of 125I-antibody/3DNA, including formulations directed to endothelial target ICAM-1, showed accurate classification of free 125I species in blood and tissues. In addition, this technique rendered data on the in vivo degradation of the traced agents over time. Thus, this is a valuable technique to obtain accurate measurements of biodistribution using 125I and possibly other radiotracers.

4.
J Control Release ; 305: 41-49, 2019 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31100312

RESUMO

DNA nanostructures hold great potential for drug delivery. However, their specific targeting is often compromised by recognition by scavenger receptors involved in clearance. In our previous study in cell culture, we showed targeting specificity of a 180 nm, 4-layer DNA-built nanocarrier called 3DNA coupled with antibodies against intercellular adhesion molecule-1 (ICAM-1), a glycoprotein overexpressed in the lungs in many diseases. Here, we examined the biodistribution of various 3DNA formulations in mice. A formulation consisted of 3DNA whose outer-layer arms were hybridized to secondary antibody-oligonucleotide conjugates. Anchoring IgG on this formulation reduced circulation and kidney accumulation vs. non-anchored IgG, while increasing liver and spleen clearance, as expected for a nanocarrier. Anchoring anti-ICAM changed the biodistribution of this antibody similarly, yet this formulation specifically accumulated in the lungs, the main ICAM-1 target. Since lung targeting was modest (2-fold specificity index over IgG formulation), we pursued a second preparation involving direct hybridization of primary antibody-oligonucleotide conjugates to 3DNA. This formulation had prolonged stability in serum and showed a dramatic increase in lung distribution: the specificity index was 424-fold above a matching IgG formulation, 144-fold more specific than observed for PLGA nanoparticles of similar size, polydispersity, ζ-potential and antibody valency, and its lung accumulation increased with the number of anti-ICAM molecules per particle. Immunohistochemistry showed that anti-ICAM and 3DNA components colocalized in the lungs, specifically associating with endothelial markers, without apparent histological changes. The degree of in vivo targeting for anti-ICAM/3DNA-nanocarriers is unprecedented, for which this platform technology holds great potential to develop future therapeutic applications.


Assuntos
DNA/metabolismo , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos , Imunoconjugados/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Animais , DNA/farmacocinética , Portadores de Fármacos/farmacocinética , Imunoconjugados/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Nanoestruturas/análise , Ratos , Distribuição Tecidual
5.
Biotechniques ; 44(6): 815-8, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18476836

RESUMO

DNA dendrimers, conjugated with both anti-biotin antibodies and up to 350 labeling entities, were designed and adapted to protein microarray and enzyme-linked immunosorbent assay (ELISA) to improve the limits of protein detection with no additional steps or equipment. Application of conjugated dendrimers to standard ELISA cytokine detection resulted in up to threefold improvement of the limits of detection with no significant increase in the inter- and intra-assay coefficient of variation (CV) compared to streptavidin horseradish peroxidase (SA-HRP) detection. The adaptation of conjugated dendrimers to protein microarray cytokine detection resulted in up to 10-fold improvement of the limits of detection, but assay conditions would have to be optimized to decrease the intra- and inter-assay %CVs.


Assuntos
DNA/química , DNA/genética , Perfilação da Expressão Gênica/métodos , Técnicas de Amplificação de Ácido Nucleico , Proteínas/análise , Proteínas/genética , RNA/genética , Dendrímeros , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
6.
Nucleic Acids Res ; 30(18): 4075-87, 2002 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-12235392

RESUMO

We find that nuclear protein extracts from mammalian cells contain an activity that allows DNA ends to associate with circular pUC18 plasmid DNA. This activity requires the catalytic subunit of DNA-PK (DNA-PKcs) and Ku since it was not observed in mutants lacking Ku or DNA-PKcs but was observed when purified Ku/DNA-PKcs was added to these mutant extracts. Purified Ku/DNA-PKcs alone did not produce association of DNA ends with plasmid DNA suggesting that additional factors in the nuclear extract are necessary for this activity. Competition experiments between pUC18 and pUC18 plasmids containing various nuclear matrix attachment region (MAR) sequences suggest that DNA ends preferentially associate with plasmids containing MAR DNA sequences. At a 1:5 mass ratio of MAR to pUC18, approximately equal amounts of DNA end binding to the two plasmids were observed, while at a 1:1 ratio no pUC18 end binding was observed. Calculation of relative binding activities indicates that DNA end-binding activities to MAR sequences was 7-21-fold higher than pUC18. Western analysis of proteins bound to pUC18 and MAR plasmids indicates that XRCC4, DNA ligase IV and scaffold attachment factor A preferentially associate with the MAR plasmid in the absence or presence of DNA ends. In contrast, Ku and DNA-PKcs were found on the MAR plasmid only in the presence of DNA ends suggesting that binding of these proteins to DNA ends is necessary for their association with MAR DNA. The ability of DNA-PKcs/Ku to direct DNA ends to MAR and pUC18 plasmid DNA is a new activity for DNA-PK and may be important for its function in double-strand break repair. A model for DNA repair based on these observations is presented.


Assuntos
Antígenos Nucleares , DNA Helicases , DNA/metabolismo , Matriz Nuclear/genética , Plasmídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência Rica em At/genética , Animais , Ligação Competitiva , Células CHO , Cricetinae , Reparo do DNA/genética , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Autoantígeno Ku , Mutação , Proteínas Nucleares/metabolismo , Plasmídeos/genética , Células Tumorais Cultivadas
7.
Cancer Res ; 76(6): 1549-59, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26921342

RESUMO

Growing evidence shows that cancer cells use mRNA-binding proteins and miRNAs to posttranscriptionally regulate signaling pathways to adapt to harsh tumor microenvironments. In ovarian cancer, cytoplasmic accumulation of mRNA-binding protein HuR (ELAVL1) is associated with poor prognosis. In this study, we observed high HuR expression in ovarian cancer cells compared with ovarian primary cells, providing a rationale for targeting HuR. RNAi-mediated silencing of HuR in ovarian cancer cells significantly decreased cell proliferation and anchorage-independent growth, and impaired migration and invasion. In addition, HuR-depleted human ovarian xenografts were smaller than control tumors. A biodistribution study showed effective tumor-targeting by a novel Cy3-labeled folic acid (FA)-derivatized DNA dendrimer nanocarrier (3DNA). We combined siRNAs against HuR with FA-3DNA and found that systemic administration of the resultant FA-3DNA-siHuR conjugates to ovarian tumor-bearing mice suppressed tumor growth and ascites development, significantly prolonging lifespan. NanoString gene expression analysis identified multiple HuR-regulated genes that function in many essential cellular and molecular pathways, an attractive feature of candidate therapeutic targets. Taken together, these results are the first to demonstrate the versatility of the 3DNA nanocarrier for in vivo-targeted delivery of a cancer therapeutic and support further preclinical investigation of this system adapted to siHuR-targeted therapy for ovarian cancer.


Assuntos
Proliferação de Células/efeitos dos fármacos , Proteína Semelhante a ELAV 1/administração & dosagem , Nanopartículas/administração & dosagem , Neoplasias Ovarianas/tratamento farmacológico , RNA Mensageiro/administração & dosagem , Proteínas de Ligação a RNA/administração & dosagem , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Sistemas de Liberação de Medicamentos/métodos , Proteína Semelhante a ELAV 1/genética , Feminino , Células HEK293 , Heterozigoto , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/administração & dosagem , MicroRNAs/genética , Neoplasias Ovarianas/genética , Interferência de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Distribuição Tecidual/genética
8.
RNA Biol ; 2(3): 93-100, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-17114923

RESUMO

MicroRNAs (miRNAs) are post-transcriptional regulators participating in biological processes ranging from differentiation to carcinogenesis. We developed a rational probe design algorithm and a sensitive labelling scheme for optimizing miRNA microarrays. Our microarray contains probes for all validated miRNAs from five species, with the potential for drawing on species conservation to identify novel miRNAs with homologous probes. These methods are useful for high-throughput analysis of micro RNAs from various sources, and allow analysis with limiting quantities of RNA. The system design can also be extended for use on Luminex beads or on 96-well plates in an ELISA-style assay. We optimized hybridization temperatures using sequence variations on 20 of the probes and determined that all probes distinguish wild-type from 2 nt mutations, and most probes distinguish a 1 nt mutation, producing good selectivity between closely-related small RNA sequences. Results of tissue comparisons on our microarrays reveal patterns of hybridization that agree with results from Northern blots and other methods.


Assuntos
MicroRNAs/análise , MicroRNAs/genética , Sondas Moleculares , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Células Cultivadas , Ratos , Ratos Long-Evans
9.
BMC Genomics ; 5: 76, 2004 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-15469607

RESUMO

BACKGROUND: RNA amplification is required for incorporating laser-capture microdissection techniques into microarray assays. However, standard oligonucleotide microarrays contain sense-strand probes, so traditional T7 amplification schemes producing anti-sense RNA are not appropriate for hybridization when combined with conventional reverse transcription labeling methods. We wished to assess the accuracy of a new sense-strand RNA amplification method by comparing ratios between two samples using quantitative real-time PCR (qPCR), mimicking a two-color microarray assay. RESULTS: We performed our validation using qPCR. Three samples of rat brain RNA and three samples of rat liver RNA were amplified using several kits (Ambion messageAmp, NuGen Ovation, and several versions of Genisphere SenseAmp). Results were assessed by comparing the liver/brain ratio for 192 mRNAs before and after amplification. In general, all kits produced strong correlations with unamplified RNAs. The SenseAmp kit produced the highest correlation, and was also able to amplify a partially degraded sample accurately. CONCLUSION: We have validated an optimized sense-strand RNA amplification method for use in comparative studies such as two-color microarrays.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , RNA Mensageiro/genética , Animais , Química Encefálica , Biblioteca Gênica , Fígado/química , Ratos
10.
Methods Mol Biol ; 822: 117-29, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22144195

RESUMO

Microarrays have been used extensively for messenger RNA expression monitoring. Recently, microarrays have been designed to interrogate expression levels of noncoding RNAs. Here, we describe methods for RNA labeling and the use of a miRNA array to identify and measure microRNA present in RNA samples.


Assuntos
Perfilação da Expressão Gênica/métodos , MicroRNAs/análise , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Biologia Computacional/métodos , Perfilação da Expressão Gênica/instrumentação , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Poli A , Controle de Qualidade , RNA/química , RNA/isolamento & purificação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos
11.
J Mol Diagn ; 14(1): 12-21, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22074760

RESUMO

Although a number of technical parameters are now being examined to optimize microRNA profiling experiments, it is unknown whether reagent or component changes to the labeling step affect starting RNA requirements or microarray performance. Human brain/lung samples were each labeled in duplicate, at 1.0, 0.5, 0.2, and 0.1 µg of total RNA, by means of two kits that use the same labeling procedure but differ in the reagent composition used to label microRNAs. Statistical measures of reliability and validity were used to evaluate microarray data. Cross-platform confirmation was accomplished using TaqMan microRNA assays. Synthetic microRNA spike-in experiments were also performed to establish the microarray signal dynamic range using the ligation-modified kit. Technical replicate correlations of signal intensity values were high using both kits, but improved with the ligation-modified assay. The drop in detection call sensitivity and miRNA gene list correlations, when using reduced amounts of standard-labeled RNA, was considerably improved with the ligation-modified kit. Microarray signal dynamic range was found to be linear across three orders of magnitude from 4.88 to 5000 attomoles. Thus, optimization of the microRNA labeling reagent can result in at least a 10-fold decrease in microarray total RNA requirements with little compromise to data quality. Clinical investigations bottlenecked by the amount of starting material may use a ligation mix modification strategy to reduce total RNA requirements.


Assuntos
Perfilação da Expressão Gênica/métodos , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Coloração e Rotulagem/métodos , Animais , Biotina/química , Encéfalo/metabolismo , Perfilação da Expressão Gênica/normas , Humanos , Pulmão/metabolismo , MicroRNAs/química , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos/normas , Kit de Reagentes para Diagnóstico , Padrões de Referência , Reprodutibilidade dos Testes , Titulometria
12.
PLoS One ; 7(2): e31241, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22359580

RESUMO

Inflammatory Bowel Disease--comprised of Crohn's Disease and Ulcerative Colitis (UC)--is a complex, multi-factorial inflammatory disorder of the gastrointestinal tract. In this study we have explored the utility of naturally occurring circulating miRNAs as potential blood-based biomarkers for non-invasive prediction of UC incidences. Whole genome maps of circulating miRNAs in micro-vesicles, Peripheral Blood Mononuclear Cells and platelets have been constructed from a cohort of 20 UC patients and 20 normal individuals. Through Significance Analysis of Microarrays, a signature of 31 differentially expressed platelet-derived miRNAs has been identified and biomarker performance estimated through a non-probabilistic binary linear classification using Support Vector Machines. Through this approach, classifier measurements reveal a predictive score of 92.8% accuracy, 96.2% specificity and 89.5% sensitivity in distinguishing UC patients from normal individuals. Additionally, the platelet-derived biomarker signature can be validated at 88% accuracy through qPCR assays, and a majority of the miRNAs in this panel can be demonstrated to sub-stratify into 4 highly correlated intensity based clusters. Analysis of predicted targets of these biomarkers reveal an enrichment of pathways associated with cytoskeleton assembly, transport, membrane permeability and regulation of transcription factors engaged in a variety of regulatory cascades that are consistent with a cell-mediated immune response model of intestinal inflammation. Interestingly, comparison of the miRNA biomarker panel and genetic loci implicated in IBD through genome-wide association studies identifies a physical linkage between hsa-miR-941 and a UC susceptibility loci located on Chr 20. Taken together, analysis of these expression maps outlines a promising catalog of novel platelet-derived miRNA biomarkers of clinical utility and provides insight into the potential biological function of these candidates in disease pathogenesis.


Assuntos
Colite Ulcerativa/diagnóstico , Estudo de Associação Genômica Ampla , MicroRNAs/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Humanos , Inflamação/imunologia , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Máquina de Vetores de Suporte
14.
PLoS One ; 6(6): e20769, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21698099

RESUMO

Effective diagnosis and surveillance of complex multi-factorial disorders such as cancer can be improved by screening of easily accessible biomarkers. Highly stable cell free Circulating Nucleic Acids (CNA) present as both RNA and DNA species have been discovered in the blood and plasma of humans. Correlations between tumor-associated genomic/epigenetic/transcriptional changes and alterations in CNA levels are strong predictors of the utility of this biomarker class as promising clinical indicators. Towards this goal microRNAs (miRNAs) representing a class of naturally occurring small non-coding RNAs of 19-25 nt in length have emerged as an important set of markers that can associate their specific expression profiles with cancer development. In this study we investigate some of the pre-analytic considerations for isolating plasma fractions for the study of miRNA biomarkers. We find that measurement of circulating miRNA levels are frequently confounded by varying levels of cellular miRNAs of different hematopoietic origins. In order to assess the relative proportions of this cell-derived class, we have fractionated whole blood into plasma and its ensuing sub-fractions. Cellular miRNA signatures in cohorts of normal individuals are catalogued and the abundance and gender specific expression of bona fide circulating markers explored after calibrating the signal for this interfering class. A map of differentially expressed profiles is presented and the intrinsic variability of circulating miRNA species investigated in subsets of healthy males and females.


Assuntos
Biomarcadores/sangue , MicroRNAs/fisiologia , Feminino , Humanos , Masculino , MicroRNAs/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Expert Rev Mol Diagn ; 7(6): 775-85, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18020907

RESUMO

Gene expression analysis has facilitated a more complete understanding of the molecular biology of cellular processes and how variations of RNA expression are useful for the classification of various diseases. Furthermore, recent analysis of a variety of noncoding RNAs, such as microRNAs, has demonstrated that these RNAs play an important role in many cellular events, including cell differentiation and death, and may also serve as biological markers for disease. Besides helping in the understanding of diseases, RNA analysis is used in drug discovery, patient prognosis and treatment evaluation. One obstacle left to overcome is the amount of material required for the analysis, particularly when trying to extract information from precious, limited, clinical samples. Here we review the many approaches scientists take to either amplify the amount of RNA or amplify the signal generated from small amounts of RNA.


Assuntos
Genômica , Técnicas de Amplificação de Ácido Nucleico , RNA/análise , Animais , Expressão Gênica , Humanos , Sondas Moleculares/genética , Sondas Moleculares/metabolismo , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/química , RNA/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
16.
Mol Cell Probes ; 20(2): 114-20, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16460913

RESUMO

Acute promyelocytic leukemia (AML-M3) is characterized by a translocation between chromosomes 15 and 17 [t(15;17)]. The detection of t(15;17) at the single cell level, is commonly done by fluorescence in situ hybridization (FISH) using recombinant locus specific genomic probes greater than 14 kilobases kb in length. To allow a more thorough study of t(15;17), we designed small (0.9-3.6 kb), target-specific, single-copy probes from the human genome sequence. A novel detection approach was evaluated using moieties possessing more fluorophores, DNA dendrimers (up to 375 fluorophores per dendrimer). Two detection approaches were evaluated using the dendrimers: (1) dendrimers modified with anti-biotin antibodies for detection of biotinylated bound probes, and (2) dendrimers modified with 45-base long oligonucleotides designed from the single-copy probes, for direct detection of the target region. The selectivity of the probes was confirmed via indirect labeling with biotin/digoxigenin by nick translation, with detection efficiencies between 50 and 90%. Furthermore, the scFISH probes were successfully detected on metaphase cells with anti-biotin dendrimer conjugates and on interphase cells with 45-base modified dendrimers. Our results bring up the possibility to detect target regions of less than 1 kb, which will be a great contribution to high-resolution analysis of genomic sequences.


Assuntos
Genoma Humano , Leucemia Promielocítica Aguda/genética , Translocação Genética , Linhagem Celular Tumoral , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 17/genética , Sondas de DNA/química , Dendrímeros/química , Humanos , Hibridização in Situ Fluorescente , Interfase , Leucemia Promielocítica Aguda/patologia , Metáfase
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