ABSTRACT
Microribonucleic acids (miRNAs) have been linked with various regulatory functions and disorders, such as cancers and heart diseases. They, therefore, present an important target for detection technologies for future medical diagnostics. We report here a novel method for rapid and sensitive miRNA detection and quantitation using surface plasmon resonance (SPR) sensor technology and a DNA*RNA antibody-based assay. The approach takes advantage of a novel high-performance portable SPR sensor instrument for spectroscopy of surface plasmons based on a special diffraction grating called a surface plasmon coupler and disperser (SPRCD). The surface of the grating is functionalized with thiolated DNA oligonucleotides which specifically capture miRNA from a liquid sample without amplification. Subsequently, an antibody that recognizes DNA*RNA hybrids is introduced to bind to the DNA*RNA complex and enhance sensor response to the captured miRNA. This approach allows detection of miRNA in less than 30 min at concentrations down to 2 pM with an absolute amount at high attomoles. The methodology is evaluated for analysis of miRNA from mouse liver tissues and is found to yield results which agree well with those provided by the quantitative polymerase chain reaction (qPCR).
Subject(s)
Biosensing Techniques/methods , MicroRNAs/analysis , Surface Plasmon Resonance/methods , Animals , Antibodies , DNA , Limit of Detection , Liver/chemistry , Methods , Mice , Nucleic Acid Hybridization/immunology , RNAABSTRACT
Hepatoid adenocarcinoma (HAC) is a special type of extrahepatic adenocarcinoma, which has a striking morphologic similarity to hepatocellular carcinoma. Seven HACs arising in the stomach and one in the lung, all with liver metastasis, were studied. They shared clinical features, such as old age, high serum alpha-fetoprotein level, aggressive behavior, and hepatic tumor in absence of risk factors for hepatocellular carcinoma (HCC). Morphologically, tumors were characterized by an admixture of tubulo-and/or papillary adenocarcinoma with hepatoid foci. In six cases, liver metastases showed an exclusive hepatoid differentiation, virtually indistinguishable from HCC with solid growth pattern. As HAC and HCC cannot be differentiated on the basis of morphology alone, differences in immunohistochemical reaction patterns would be of considerable diagnostic help. Immunostaining for CK7, CK8, CK18, CK19, CK20, alpha-fetoprotein, p-CEA, and HepPar1 revealed that hepatoid areas of both primary and metastatic HAC have a specific immunoprofile, distinctive of this entity. On the one hand, positivity of virtually all HACs for alpha-fetoprotein, CK8, CK18, and the membranous, canalicular staining for polyclonal carcinoembryonic antigen underline its hepatoid nature. On the other hand, positive staining for CK19 and CK20 and frequent negativity for HepPar1 in both primary tumors and their metastases were distinctive features of HAC. Furthermore, HAC differs from combined hepatocellular cholangiocarcinoma, being negative for CK7. In addition, for comparison of immunohistochemical results, we stained with the same antibody panel a tissue microarray of 121 HCCs. Comparative genomic hybridization study of three HAC supports their hepatoid differentiation as aberrations found in HAC are common in HCC (4q-, 8p-), and hepatoblastoma (Xq+), respectively.
Subject(s)
Adenocarcinoma/diagnosis , Antibodies, Monoclonal/immunology , Biomarkers, Tumor/immunology , Carcinoma, Hepatocellular/diagnosis , Liver Neoplasms/diagnosis , Lung Neoplasms/pathology , Stomach Neoplasms/pathology , Adenocarcinoma/genetics , Adenocarcinoma/immunology , Adenocarcinoma/secondary , Aged , Diagnosis, Differential , Female , Humans , Immunohistochemistry/methods , Liver Neoplasms/genetics , Liver Neoplasms/immunology , Liver Neoplasms/secondary , Lung Neoplasms/genetics , Lung Neoplasms/immunology , Male , Middle Aged , Nucleic Acid Hybridization/genetics , Nucleic Acid Hybridization/immunology , Nucleic Acid Hybridization/methods , Stomach Neoplasms/genetics , Stomach Neoplasms/immunologyABSTRACT
BACKGROUND: Array-comparative genomic hybridization (CGH), although providing much higher resolution compared with conventional CGH, has not yet become a widely applied method for the analysis of genomic gains and losses. METHODS: In January 2002, the Wellcome Trust sponsored a workshop where many of the laboratories developing this technology met to compare different methodologies for array-CGH. Fourteen groups participated, comprising 11 from Europe and 3 from the United States. To facilitate objective analysis, each laboratory constructed arrays using the same anonymous clones and performed a series of test hybridizations using identical genomic DNAs. RESULTS: A figure of merit (FM) was developed to summarize entire collections of data from each laboratory in a single measurement. The FMs consistently showed that a few groups produced quantitative array hybridization data of high quality, whereas a majority achieved a lower standard. CONCLUSIONS: The conclusions of the workshop were that polymerase chain reaction-based methods for the amplification of large insert clones for arraying were effective for array-CGH. It was also concluded that hybridizations performed under coverslips or in automated hybridization apparatus were less effective than hybridizations performed in simple wells with gentle rocking. A common experience by the participants was the batch-to-batch variability of commercial Cot1 preparations in their ability to suppress hybridization to repeat sequences. (Supplementary material for this article can be found in the online issue, which is available at http://www.interscience.wiley.com/jpages/0196-4763/suppmat/49_2/v49.43.html or at http://www.sanger.ac.uk/HGP/Cytogenetics/Publications/Cytometry Sept 2002/Supplemental.pdf.)
Subject(s)
Genetics, Medical/trends , Genomics , Nucleic Acid Hybridization/methods , Oligonucleotide Array Sequence Analysis/methods , Technology Assessment, Biomedical , Animals , Drosophila/genetics , Education , Female , Humans , Male , Nucleic Acid Hybridization/immunology , Polymerase Chain ReactionABSTRACT
No disponible
Subject(s)
Molecular Biology/methods , Lymphoma, Non-Hodgkin/diagnosis , Lymphoma, Non-Hodgkin/etiology , Lymphoma, Non-Hodgkin/pathology , DNA Mutational Analysis/methods , Fluorescent Antibody Technique/methods , Nucleic Acid Hybridization/immunology , Nucleic Acid Hybridization/methods , Nucleic Acid Hybridization/physiology , Translocation, Genetic/genetics , Translocation, Genetic/immunology , Translocation, Genetic/physiology , Polymerase Chain Reaction/methods , Polymerase Chain Reaction , Chromosome Aberrations/genetics , Chromosome Aberrations/immunology , DNA, Neoplasm/analysisABSTRACT
One component of the B cell activation cascade is the induction of the protooncogene c-fos. Data presented here demonstrate that stimulation of mIgM-bearing cells with either anti-IgM or the combination of PDB plus ionomycin generated comparable levels of c-fos RNA. Furthermore, a synergistic response was observed when the cells were treated with selected concentrations of anti-IgM plus either PDB or ionomycin. In contrast, stimulation of mIgG-bearing B cells with anti-IgG did not induce the c-fos RNA levels that were seen when these cells were treated with the combination of PDB plus ionomycin. Treatment of mIgG-bearing cells with only the combination of anti-IgG plus ionomycin yielded a synergistic response and anti-IgG plus PDB did not. Thus, induction of c-fos RNA appears to be different in mIgM- and mIgG-bearing B cells after stimulation through mIg.
