RESUMO
The advent of automated systems for gene expression profiling has accentuated the need for the development of convenient and cost-effective methods for reagent preparation. We have developed a method for the preparation and storage of pre-aliquoted cocktail plates that contain all reagents required for amplification of nucleic acid by reverse transcription and in vitro transcription reactions. Plates can be stored at -80 degrees C for at least 1 month and kept in a hotel at 4 degrees C for at least 24 h prior to use. Microarray data quality generated from these pre-aliquoted reagent plates is not statistically different between cRNA amplified with stored cocktails and cRNA amplified with freshly prepared cocktails. Deployment of pre-aliquoted, stored cocktail plates in a fully automated system not only increases the throughput of amplifying cRNA targets from thousands of RNA samples, but could also considerably reduce reagent costs and potentially improve process robustness.
Assuntos
Hibridização Genética , Indicadores e Reagentes/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Automação , Custos e Análise de Custo , DNA Complementar/análise , Interpretação Estatística de Dados , Humanos , Células Jurkat , Células K562 , Análise de Sequência com Séries de Oligonucleotídeos/economia , RNA Complementar/análise , Transcrição ReversaRESUMO
SS18-SSX fusion genes resulting from a chromosomal translocation t(X;18)(p11.2;q11.2) are a genetic hallmark of synovial sarcoma. Although such cytogenetic or molecular aberrations have mostly been detected by fluorescence in situ hybridization or reverse transcription-polymerase chain reaction, the expression of SS18-SSX has been poorly investigated at a cellular or tissue level. In this study, biotinylated tyramide (BT)-based in situ hybridization (ISH) was performed to detect SS18-SSX transcripts using formalin-fixed, paraffin-embedded tissues from 15 synovial sarcomas. Digoxigenin-labeled cRNA probes flanking the fusion points of SS18-SSX1 and SS18-SSX2 were generated by in vitro transcription, and hybridized signals were detected by a streptavidin-biotin complex method after chemical enhancement with BT. The localizations of signals were compared with the immunohistochemical expressions of epithelial or neuroectodermal markers and those of cell adhesion including cytokeratins (CAM5.2, AE1/AE3, CK7), epithelial membrane antigen, E-cadherin, beta-catenin, c-erbB-2 (HER2/neu), CD56, and claudin-1. The ISH signals of the SS18-SSX transcripts were identified in 13 synovial sarcomas, and their fusion types correlated with those determined by reverse transcription-polymerase chain reaction. In biphasic tumors, the ISH signals tended to localize to epithelial areas, whereas spindle-cell areas or monophasic fibrous tumors showed a less intense or focal expression pattern. Notably, the expression patterns of AE1/AE3, CK7, and c-erbB-2 often colocalized with the ISH signals (7 of 11 cases positive for each marker). Our results suggest that BT-based ISH can be used as a molecular technique for the detection of SS18-SSX using formalin-fixed, paraffin-embedded tissues.
Assuntos
Proteínas de Neoplasias/genética , Fusão Oncogênica , Proteínas Proto-Oncogênicas/genética , RNA Complementar/análise , Proteínas Repressoras/genética , Sarcoma Sinovial/genética , Transcrição Gênica , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/análise , Feminino , Formaldeído/química , Humanos , Hibridização In Situ/métodos , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Sondas RNA/química , Sarcoma Sinovial/química , Sarcoma Sinovial/patologiaRESUMO
A tetrakis-acridinyl peptide (TAP) cassette, consisting of a double-stranded region of alternating AT sequence bound to TAP and a single stranded overhanging sequence of continuous dA, was prepared by mixing TAP with d[A18(TA)51]. A TAP cassette could be applied to the fluorometric detection of hybridized DNA on the DNA chip, which was prepared by stamping a 45-meric DNA probe onto a gold-coated plastic chip using a high-precision spotter developed at RIKEN. Spots on the DNA chip were imaged by a CCD camera after hybridization with 65-meric target single-stranded DNAs carrying a continuous dA20 sequence (dA tail) on the DNA chip after treatment with a TAP cassette. Their fluorescence intensity on the DNA chip showed a good linear correlation with the concentration of the target DNAs in the range from 10 pM to 1 nM. Fluorescence of their spots derived from the TAP cassette remaining on the surface of the DNA chip through the dA tail of the hybridized target DNA. Furthermore, the TAP cassette could be successfully applied to the quantitative detection of complementary RNAs (cRNAs) prepared from rat brain with reverse transcription and in vitro transcription.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/genética , RNA Complementar/análise , RNA Complementar/genética , Animais , Sequência de Bases , RatosRESUMO
We developed a bispyrene-conjugated 2'-O-methyloligoribonucleotide as an RNA-specific RNA-probe. The probe hybridized with the complementary RNA, greatly enhancing fluorescence and discriminating RNA from DNA. The assay was carried out in homogeneous aqueous media without removing the unbound probe from the detection solution. This homogeneous fluorescence assay also discriminated mismatch sequences in the target RNA. These pyrene probes could possess high potential to detect RNA in biological specimens simply.
Assuntos
Citidina/análogos & derivados , Hibridização de Ácido Nucleico , Sondas RNA/química , RNA Complementar/análise , Espectrometria de Fluorescência/métodos , Uridina/análogos & derivados , Citidina/síntese química , Citidina/química , Metilação , Oligorribonucleotídeos/química , Pirenos/química , Uridina/síntese química , Uridina/químicaRESUMO
Small RNAs (sRNAs) are short (18-30 nucleotide) noncoding RNA molecules, which control gene expression and pathogen response in eukaryotes. They are associated with and guide nucleases to target nucleic acids by nucleotide base pairing. We found that current techniques for small RNA detection are adversely affected by the presence of complementary RNA. Thus we established FDF-PAGE (fully denaturing formaldehyde polyacrylamide gel electrophoresis), which dramatically improves denaturation efficiency and subsequently the detection of sequestered sRNAs.
Assuntos
Northern Blotting/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Formaldeído/química , Desnaturação de Ácido Nucleico , Pequeno RNA não Traduzido/análise , Pareamento de Bases , Hibridização de Ácido Nucleico/métodos , RNA Complementar/análise , Ureia/químicaRESUMO
Microarray analysis is a powerful technique for high-throughput, global transcriptonomic profiling of gene expression. It holds great promise for analyzing the genetic and molecular bases of cardiovascular diseases and various other complex diseases and permits the analysis of thousands of genes simultaneously, both in diseased and nondiseased tissues and/or cell lines. Microarrays or microchips are made by depositing spots of DNA or oligonucleotides representing thousands of genes on a solid support such as a coated glass surface, and can allow the comparison of gene expression patterns in any two samples. Total RNA is isolated from the tissue or cells of interest, converted to cDNA and then cRNA labeled with biotin, and hybridized to the chips. Hybridization signals are then quantified and compared among different samples. We used oligonucleotide microarrays to obtain an unbiased assessment of expression levels of thousands of genes simultaneously in normal and diseased coronary arteries. Fifty-six genes showed differential expression in atherosclerotic coronary artery tissues, and 49 of them represent new linked genes for coronary artery disease. These studies can generate novel hypotheses relating to the pathologies of disease and further studies with animal models, molecular biology, cell biology, and biochemistry will validate these hypotheses and provide novel insights into the pathogenesis of disease.
Assuntos
Doenças Cardiovasculares/metabolismo , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Biotina , Doenças Cardiovasculares/genética , DNA/isolamento & purificação , Sondas de DNA/síntese química , Humanos , RNA Complementar/análise , RNA Complementar/síntese químicaRESUMO
Direct labeling of RNA is an expedient method for labeling large quantities (e.g. micrograms) of target RNA for microarray analysis. We have developed an efficient labeling system that uses T4 RNA ligase to attach a 3'-biotinylated donor molecule to target RNA. Microarray analyses indicate that directly labeled RNA is uniformly labeled, has higher signal intensity than comparable labeling methods and achieves high transcript detection sensitivity. The labeled donor molecule we have developed allows the attachment of multiple biotins, which increases target signal intensity up to 30%. We have used this direct-labeling method to detect previously discovered class predictor genes for two types of cancer: acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In order to test the sensitivity of direct RNA labeling, we analyzed the AML and ALL expression profiles for predictor genes that were previously found to show elevated expression in the disease state. Direct labeling of AML poly(A) RNA detects 90% of the class predictor genes that are detected by the IVT-based target amplification method used to discover the genes. These results indicate that the detection sensitivity, simplicity (single tube reaction) and speed (2 h) of this direct labeling protocol may be ideal for diagnostic applications that do not require target amplification.
Assuntos
Biotinilação , Perfilação da Expressão Gênica/métodos , Leucemia/classificação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Neoplásico/análise , Doença Aguda , Linhagem Celular Tumoral , Humanos , Leucemia/diagnóstico , Leucemia/genética , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Oligorribonucleotídeos/biossíntese , Oligorribonucleotídeos/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RNA Complementar/análise , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismoRESUMO
The peroxisome proliferator activated receptor PPAR-gamma has been identified as a nuclear receptor for thiazolidenediones, which are compounds with insulin-sensitizing properties in several tissues, including skeletal muscle. To determine whether this receptor is expressed and possibly involved in insulin action/resistance in skeletal muscle, PPAR-gamma mRNA abundance and its regulation by insulin were quantified in muscle tissue and cultures from lean and obese nondiabetic and type II diabetic subjects using competitive reverse transcription-polymerase chain reaction (RT-PCR). In muscle biopsy specimens, PPAR-gamma mRNA was elevated in obese nondiabetic and type II diabetic subjects (23.4 +/- 4.2 and 28.0 +/- 5.69 x 10(3) copies/microg total RNA, respectively; both P < 0.05) compared with lean nondiabetic control subjects (9.4 +/- 2.3 x 10(3) copies/microg total RNA). Significant positive correlations were present among skeletal muscle PPAR-gamma mRNA levels, BMI (r = 0.67, P < 0.01), and fasting insulin concentration (r = 0.76, P < 0.001). PPAR-gamma mRNA levels were also elevated in muscle cultures from type II diabetic subjects compared with lean nondiabetic control subjects (330.1 +/- 52.9 vs. 192.1 +/- 27.0 x 10(3) copies/microg total RNA, P < 0.05). Insulin stimulation of muscle tissue (by hyperinsulinemic-euglycemic clamp for 3-4 h) or muscle cultures (30 nmol/l for 120 min) stimulated PPAR-gamma mRNA expression up to fourfold (10.0 +/- 2.7 to 41.3 +/- 7.4 x 10(3) copies/microg total RNA, P < 0.05, and 174.9 +/- 56.9 to 268.2 +/- 78.6 x 10(3) copies/microg total RNA, P < 0.05, respectively). In summary, PPAR-gamma mRNA expression in human skeletal muscle is acutely regulated by insulin and is increased in both obese nondiabetic and type II diabetic subjects in direct relation to BMI and fasting insulinemia. We conclude that abnormalities of PPAR-gamma may be involved in skeletal muscle insulin resistance of obesity and type II diabetes.
Assuntos
Diabetes Mellitus Tipo 2/genética , Expressão Gênica/genética , Músculo Esquelético/fisiologia , Proteínas Nucleares/genética , Obesidade/genética , RNA Mensageiro/análise , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Adulto , Índice de Massa Corporal , Diabetes Mellitus Tipo 2/complicações , Jejum , Expressão Gênica/efeitos dos fármacos , Humanos , Insulina/sangue , Insulina/farmacologia , Pessoa de Meia-Idade , Músculo Esquelético/efeitos dos fármacos , Reação em Cadeia da Polimerase , RNA Complementar/análise , RNA Complementar/genética , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacosRESUMO
The central neuropeptide Y (NPY) Y1 receptor (Y1-R) system has been implicated in feeding, endocrine, and autonomic regulation. In the present study, we systematically examined the brain distribution of Y1-R mRNA in rodents by using radioisotopic in situ hybridization histochemistry (ISHH) with a novel sensitive cRNA probe. Within the rat hypothalamus, Y1-R-specific hybridization was observed in the anteroventral periventricular, ventromedial preoptic, suprachiasmatic, paraventricular (PVH), dorsomedial, ventromedial, arcuate, and mamillary nuclei. In the rat, Y1-R mRNA expression was also seen in the subfornical organ, anterior hypothalamic area, dorsal hypothalamic area, and in the lateral hypothalamic area. In addition, Y1-R hybridization was evident in several extrahypothalamic forebrain and hindbrain sites involved in feeding and/or autonomic regulation in the rat. A similar distribution pattern of Y1-R mRNA was observed in the mouse brain. Moreover, by using a transgenic mouse line expressing green fluorescent protein under the control of the melanocortin-4 receptor (MC4-R) promoter, we observed Y1-R mRNA expression in MC4-R-positive cells in several brain sites such as the PVH and central nucleus of the amygdala. Additionally, dual-label ISHH demonstrated that hypophysiotropic PVH cells coexpress Y1-R and pro-thyrotropin-releasing hormone mRNAs in the rat. These observations are consistent with the proposed roles of the central NPY/Y1-R system in energy homeostasis.
Assuntos
Mapeamento Encefálico , Hipotálamo/metabolismo , RNA Mensageiro/metabolismo , Receptor Tipo 4 de Melanocortina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Animais , Regulação do Apetite/fisiologia , Comportamento Alimentar/fisiologia , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Transgênicos , Prosencéfalo/metabolismo , RNA Complementar/análise , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/genética , Rombencéfalo , Distribuição TecidualRESUMO
Gene expression profiling from microdissected cell populations is a powerful approach to explore molecular processes involved in development and solid tumor biology. In this chapter, we detail robust and validated methods for tissue preparation and isolation of high-quality RNA from microdissected cell populations. A protocol is also provided for linear transcript amplification using as little as 10 ng of total RNA to produce labeled cRNA targets for hybridization to GeneChip high-density oligonucleotide microarrays. Particular emphasis is placed on troubleshooting each technical step in the protocol and measures of quality assurance for both RNA isolation and resulting microarray data.
Assuntos
Perfilação da Expressão Gênica/métodos , Lasers , Microdissecção/métodos , Neoplasias/genética , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Complementar/análise , RNA Neoplásico/análise , Animais , Humanos , Neoplasias/química , RNA Complementar/isolamento & purificação , RNA Neoplásico/isolamento & purificaçãoRESUMO
BACKGROUND: The usefulness of log2 transformation for cDNA microarray data has led to its widespread application to Affymetrix data. For Affymetrix data, where absolute intensities are indicative of number of transcripts, there is a systematic relationship between variance and magnitude of measurements. Application of the log2 transformation expands the scale of genes with low intensities while compressing the scale of genes with higher intensities thus reversing the mean by variance relationship. The usefulness of these transformations needs to be examined. RESULTS: Using an Affymetrix GeneChip dataset, problems associated with applying the log2 transformation to absolute intensity data are demonstrated. Use of the spread-versus-level plot to identify an appropriate variance stabilizing transformation is presented. For the data presented, the spread-versus-level plot identified a power transformation that successfully stabilized the variance of probe set summaries. CONCLUSION: The spread-versus-level plot is helpful to identify transformations for variance stabilization. This is robust against outliers and avoids assumption of models and maximizations.
Assuntos
Gráficos por Computador , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Biologia Computacional/métodos , DNA Complementar/análise , Regulação da Expressão Gênica , Genes , Humanos , Processamento de Imagem Assistida por Computador/métodos , RNA Complementar/análiseRESUMO
BACKGROUND: The development of large-scale gene expression profiling technologies is rapidly changing the norms of biological investigation. But the rapid pace of change itself presents challenges. Commercial microarrays are regularly modified to incorporate new genes and improved target sequences. Although the ability to compare datasets across generations is crucial for any long-term research project, to date no means to allow such comparisons have been developed. In this study the reproducibility of gene expression levels across two generations of Affymetrix GeneChips (HuGeneFL and HG-U95A) was measured. RESULTS: Correlation coefficients were computed for gene expression values across chip generations based on different measures of similarity. Comparing the absolute calls assigned to the individual probe sets across the generations found them to be largely unchanged. CONCLUSION: We show that experimental replicates are highly reproducible, but that reproducibility across generations depends on the degree of similarity of the probe sets and the expression level of the corresponding transcript.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Calibragem , Biologia Computacional/instrumentação , Biologia Computacional/métodos , Biologia Computacional/normas , Biologia Computacional/estatística & dados numéricos , DNA Complementar/análise , DNA Complementar/genética , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/normas , Perfilação da Expressão Gênica/estatística & dados numéricos , Regulação da Expressão Gênica/genética , Humanos , Músculo Esquelético/química , Músculo Esquelético/citologia , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/normas , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , RNA/análise , RNA/genética , RNA/normas , Sondas RNA/genética , Sondas RNA/normas , RNA Complementar/análise , Distribuições EstatísticasRESUMO
Accurate quantification of mRNA by competitive RT-PCR demands that the quality of the cRNA internal standard be strictly controlled and that at least two criteria should be satisfied. First, genomic DNA should be removed from the total RNA being analyzed; second, template DNA should be removed from the cRNA internal standard following in vitro transcription. We observed that the routine use of RNase-free DNase I is insufficient for removing template DNA from cRNA samples and can degrade cRNA. Furthermore, reducing the template DNA before digestion, selectively extracting template DNA, and gel fractionation are all ineffective at completely eliminating template DNA contamination in cRNA standards. A strategy was developed ("inverted" competitive RT-PCR) to quantify template DNA contamination in cRNA standards. Regardless of treatment method, a small percentage of DNA contamination remained in the products of in vitro transcription. Without correction, the number of mRNA copies calculated from competitive RT-PCR is systematically overestimated. The number of template DNAs contaminating the cRNA samples was remarkably large, though as a percentage of the total cRNA, DNA contamination was small and could be easily corrected.
Assuntos
DNA/análise , RNA Complementar/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , DNA/metabolismo , Desoxirribonucleases/metabolismo , Padrões de ReferênciaRESUMO
In histochemical work with digoxigenin- or biotin-labeled nucleic acid probes, reproducibility of in situ hybridization depends on accurate measurement of the amount of non-radioactive label being used. We describe a rapid and sensitive assay for nonradioactive label incorporated into nucleic acids employing a luminogenic substrate for alkaline phosphatase, CSPD (disodium 3-(4-methoxyspirol¿1,2-dioxetane-3,2'-(5'-chloro)tricyclo [3.3.1.1(3,7)]decan¿-4-yl)phenyl phosphate). An alkaline phosphatase-antibody conjugate was bound to digoxigenin-labeled nucleic acids spotted on nylon membranes. Light emission from the reaction of the bound alkaline phosphatase with CSPD was measured with a luminometer. This method allows an accurate determination of digoxigenin incorporated into nucleic acid probes in the range of 0.5-500 fmol of nonradioactive label.
Assuntos
Adamantano/análogos & derivados , Sondas de DNA/análise , Digoxigenina/análise , Adamantano/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Bovinos , Medições Luminescentes , RNA Complementar/análise , Esteroide 17-alfa-Hidroxilase/genética , Fatores de TempoRESUMO
In estimations of mRNA copy numbers, quantitative in situ hybridization (ISH) is expected to be performed at saturating probe concentration. In practice, however, this condition can rarely be fulfilled when medium to high amounts of transcripts exist and/or in large-scale experiments. To resolve this problem, we developed and tested a double-step procedure involving the use of calmodulin (CaM) I, II, and III [(35)S]-cRNA probes on adult rat brain sections; the hybridization signals were detected with a phosphorimager. By means of hybridization at increasing probe concentrations for a time sufficient for saturation, saturation curves were created for and maximal hybridization capacity (Hmax) values were assigned to selected brain areas. The Kd values of these various brain areas did not differ significantly, which allowed the creation and use of one calibration graph of Hmax vs hybridized [(35)S]-cRNA values for all brain areas for a given probe concentration. Large-scale ISH experiments involving a subsaturating probe concentration were performed to estimate Hmax values for multiple CaM mRNAs. A calibration graph corresponding to this probe concentration was created and Hmax values (expressed in ISH copy no/mm(2) units) were calculated for several brain regions via the calibration. The value of the method was demonstrated by simultaneous quantification of the total accessible multiple CaM mRNA contents of several brain areas in a precise and economical way.
Assuntos
Calmodulina/genética , Hibridização In Situ/métodos , RNA Mensageiro/análise , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Calmodulina/metabolismo , DNA/análise , Masculino , RNA Complementar/análise , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Ku70 and Ku80 proteins are responsible for the repair of DNA double-strand breaks and function as a regulatory subunit of the DNA-dependent protein kinase. In this study we analyzed expression of both genes in malignant melanoma tissue arrays applying in situ hybridization probes produced by our research group and using immunohistochemical analysis. Expression of both genes was down-regulated as melanoma progressed. In situ hybridization demonstrated more Ku70- and Ku80-positive cells than immunohistochemical methods, but the correlation between the two methods was highly significant (P <0.01). We conclude that the in situ hybridization assay for the detection of Ku70 and Ku80 expression used in this study is also suitable for tissue microarray analysis of paraffin-embedded melanoma samples. The laboratory procedure is much more complicated than the immunohistochemical method, however.
Assuntos
Antígenos Nucleares/análise , Biomarcadores Tumorais/análise , DNA Helicases , Proteínas de Ligação a DNA/análise , Imuno-Histoquímica , Hibridização In Situ/métodos , Melanoma/química , Melanoma/patologia , Análise Serial de Proteínas , Adulto , Idoso , Idoso de 80 Anos ou mais , Reparo do DNA , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Autoantígeno Ku , Masculino , Melanoma/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Parafina , Reação em Cadeia da Polimerase , Sondas RNA , RNA Complementar/análise , RNA Neoplásico/análise , Neoplasias Cutâneas/química , Neoplasias Cutâneas/patologiaRESUMO
PURPOSE: Colorectal cancer is one of the most common malignancies. Substaging of the cancer is of importance not only to prognosis but also to treatment. Classification of substages based on DNA microarray technology is currently the most promising approach. We therefore investigated if gene expression microarrays could be used to classify colorectal tumors. METHODS: We used the Affymetrix oligonucleotide arrays to analyze the expression of more than 5,000 genes in samples from the sigmoid and upper rectum of the left colon. Five samples were from normal mucosa and five samples from each of the Dukes' stages A, B, C, and D. Expression data were filtered based on either covariance or a selection of the most significantly varying genes between tumor stages. RESULTS: A nearest neighbor classifier was used to classify normal, and Dukes' B and C samples with less than 20% error, whereas Dukes' A and D could not be classified correctly. A number of interesting gene clusters showed a discriminating difference between Dukes' B and C samples. These included mitochondrial genes, stromal remodeling genes, and genes related to cell adhesion. CONCLUSION: Molecular classification based on gene expression of one of the most common malignancies, colorectal cancer, now seems to be within reach. The data indicates that it is possible at least to classify Dukes' B and C colorectal tumors with microarrays.
Assuntos
Adenocarcinoma/classificação , Adenocarcinoma/genética , Neoplasias do Colo/classificação , Neoplasias do Colo/genética , Análise de Sequência com Séries de Oligonucleotídeos , Adenocarcinoma/patologia , Análise por Conglomerados , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Estadiamento de Neoplasias , RNA Complementar/análise , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We evaluated the expression of MDR1/p-glycoprotein in paediatric tumours using reverse transcriptase polymerase chain reaction (RT-PCR), RNA dot blot analysis, and immunohistochemistry on formalin fixed paraffin-embedded material with JSB-1 and C-219 monoclonal antibodies, and compared these three techniques. The expression of multidrug resistance-associated protein (MRP) gene was examined by RT-PCR assay. We studied MDR1/p-glycoprotein and MRP expression in 13 samples from 10 neuroblastoma patients, 11 samples from 10 nephroblastoma patients, 2 rhabdomyosarcomas, 1 adrenocortical carcinoma and 10 benign tumours or tumour-like lesions. Eleven of 13 neuroblastomas, 7 of 11 nephroblastomas, 2 rhabdomyosarcomas, 1 adrenocortical carcinoma, and 7 of 10 benign tumours or tumour-like lesions showed MDR1 PCR products. By RNA dot blot analysis, MDR1 transcripts were detectable in 11 of 34 specimens. Immunohistochemically, we detected positive reaction products for JSB-1 in 26 of 36 samples. There was a significant correlation between the immunoreactivity for JSB-1 and the expression of MDR1 mRNA expression by RT-PCR (P = 0.0001). However, the presence of p-glycoprotein immunostaining does not correlate with the MDR1 expression shown by RT-PCR in every case. As for MRP mRNA expression, 9 of 13 neuroblastomas and 10 of 11 nephroblastomas revealed PCR products.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Resistência a Múltiplos Medicamentos/genética , Genes MDR , Neoplasias/genética , Neoplasias/metabolismo , Transportadores de Cassetes de Ligação de ATP/biossíntese , Adolescente , Anticorpos Monoclonais/análise , Sequência de Bases , Criança , Pré-Escolar , Feminino , Humanos , Immunoblotting/métodos , Imuno-Histoquímica/métodos , Lactente , Recém-Nascido , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Neoplasias/química , Neoplasias/patologia , Reação em Cadeia da Polimerase/métodos , RNA Complementar/análise , RNA Neoplásico/análise , Sensibilidade e Especificidade , Análise de Sobrevida , Células Tumorais CultivadasRESUMO
OBJECTIVE: To evaluate maternal serum activin A levels in pregnant women at parturition, correlated to the mode of delivery, and to localize activin receptor messenger RNA in human placenta and fetal membranes. METHODS: A specific two-site enzyme-linked immunosorbent assay (ELISA) was used to measure maternal activin A levels. Activin receptor mRNA was localized in placenta and fetal membranes by in situ hybridization, using ActRII or ActRIIB antisense riboprobes. RESULTS: Serum activin A levels increased significantly in pregnant women during vaginal or cesarean delivery after spontaneous labor. No significant changes of serum activin A were found in patients undergoing elective cesarean delivery. Syncytiotrophoblast and amnion cells hybridized to radiolabeled ActRIIB probe, whereas few cells within the structure of the villi and decidual cells hybridized to radiolabeled ActRII probe. CONCLUSION: The present studies indicate that vaginal or cesarean delivery following spontaneous labor is characterized by increased activin A levels and that activin receptors are present on trophoblast and fetal membranes.
Assuntos
Vilosidades Coriônicas/metabolismo , DNA Complementar/análise , Decídua/metabolismo , Membranas Extraembrionárias/metabolismo , Inibinas/sangue , Trabalho de Parto/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Sondas RNA/análise , Receptores de Fatores de Crescimento/biossíntese , Receptores de Ativinas , Ativinas , Adulto , Sítios de Ligação , Decídua/citologia , Membranas Extraembrionárias/citologia , Feminino , Humanos , Inibinas/metabolismo , Gravidez , RNA Complementar/análise , RNA Mensageiro/metabolismoRESUMO
The present study was designed to evaluate the expression of dopamine D1 and D2 receptor mRNAs in systemic and pulmonary vasculatures. Using specific antisense riboprobes for dopamine D1 and D2 receptor cDNAs, in situ hybridization histochemistry was performed in the aorta, common carotid artery, vertebral artery, pulmonary artery, and superior vena cava of the adult male Sprague Dawley rat. In the case of the aorta, common carotid artery, and vertebral artery, dopamine D1 receptor mRNAs localized mainly in the smooth muscle cells of the tunica media. However, the signals of dopamine D2 receptor mRNAs were found in the endothelium and subendothelial layer of tunica intima, and interstitial cells of tunica adventitia. In the case of the pulmonary artery, signals of dopamine D1 receptor mRNAs were detected within the tunica intima, media, and adventitia. Expression of D2 receptor mRNAs was detected in the walls of small blood vessels within the tunica adventitia of the pulmonary artery. There were no detectable signals of dopamine D1 and D2 receptor mRNAs in the vein. The uneven distribution of dopamine D1 and D2 receptor mRNAs in the rat systemic vasculatures and pulmonary artery suggests that dopamine differentially regulates the vasodilation of the systemic and pulmonary arteries through the differential stimulation of dopamine D1 and D2 receptor.