The application of 3D micropatterning of agarose substrate for cell culture and in situ comet assays.

We report the fabrication of a 3D micropatterned agarose substrate that enables the culture of single or multiple cells. Patterning was performed on dried agarose using deep UV irradiation leading to 6-microm-deep micropatterns of 25-70 microm in diameter. Cell adhesion was facilitated by the specific grafting of ECM (extra cellular matrix) proteins such as fibronectin into the micropatterns.

2D and 3D cell microarrays in pharmacology.

To analyze the phenotypic consequences of perturbing mammalian cells with drugs, there is an increasing need for systematic cell-based assays in an HTS format. Cell microarrays provide an attractive solution as they offer more than a simple miniaturization and mechanization of conventional microtiter plates. While standard monolayer two-dimensional culture conditions are poor mimics of the cellular environment in situ, microfabricated systems enable three-dimensional organotypic cell cultures and have the potential to provide biological insight not achievable before.

Improvement of yeast biochip sensitivity using multilayer inorganic sol-gel substrates.

Today, microarray fluorescence detection is still limited because a great proportion of hybrids remain undetectable. In this paper we describe sol-gel optical multilayers (stacks of low- and high-index layers) deposited on glass slides which increase the fluorescence of DNA microarrays and favour the detection of fluorescent targets. An alternative to the expensive and time-consuming physical vapour deposition technology is proposed.

Photochemical patterning of biological molecules inside a glass capillary.

A simple way for photochemical patterning of biological molecules onto the inner wall of fused-silica capillary is described. The method is based on a modification of the inner capillary surface with photoactive benzophenone (BP) derivative. The UV irradiation at 365 nm of the capillary filled with a sample solution results in cross-linking of the solutes to the BP moiety via a stable covalent bond.

Multiple wavelength fluorescence enhancement on glass substrates for biochip and cell analyses.

In microarrays experiments, a serious limitation is the unreliability of low signal intensities data and the lack of reproducibility for the resulting ratios between samples and controls. Most of the light emitted by a fluorophore at the air/glass interface of a glass slide is absorbed by the glass so just a part of the emitted fluorescence is detected. To improve the sensitivity of the fluorescence detection of both common fluorophores Cy3 and Cy5 in DNA microarrays and fluorescent cell analyses, we have designed a multi layer mirror with alternative thin layers of SiO2 and HfO2.

Determination of surface-bound-fluorophore orientation by goniometric fluorescence polarization: application to quantification of DNA-chip readouts.

We present a polarized goniofluorimeter designed to measure the observation-angle and polarization-dependent intensity emitted by a group of surface-bound fluorescent molecules. We studied two types of surface bonding: In one case, dyes were adsorbed into the surface by spin coating, and in the other, dyes were covalently immobilized to DNA strands. Fluorescent dyes consisted of Cy3 and Alexa546.

Direct in situ reverse transcriptase-linked polymerase chain reaction with biotinylated primers for the detection of hepatitis C virus RNA in liver biopsies.

To assess the presence and the cellular distribution of hepatitis C virus (HCV) RNA in the liver of 11 patients with confirmed HCV infection, a direct in situ reverse transcriptase-linked polymerase chain reaction (RT-PCR) method was performed on formalin-fixed and paraffin-embedded biopsies. The oligonucleotide primers used were specific to the 5' non coding region. An unlabelled downstream oligonucleotide served as a primer for reverse transcription as well as PCR.

Polypyrrole DNA chip on a silicon device: example of hepatitis C virus genotyping.

We describe in this article an oligonucleotide array constructed on a silicon device bearing a matrix of addressable 50-microns microelectrodes. Each electrode was covered by a conducting polymer (polypyrrole) grafted by an oligonucleotide (ODN). The DNA chip was prepared by successive electrochemically addressed copolymerizations of 5' pyrrole-labeled ODN and pyrrole.

Rapid diagnosis of Mycobacterium tuberculosis infections by an ELISA-like detection of polymerase chain reaction products.

A polymerase chain reaction (PCR) assay was developed for the detection in clinical samples of mycobacteria belonging to the Mycobacterium tuberculosis complex. PCR products were detected with a simple and rapid colormetric method. With this method, 50 fg of M.

Detection of HIV1 DNA in biological samples by an homogeneous assay: fluorescence measurement of double-stranded RNA synthesized from amplified DNA.

A nonisotopic homogeneous detection of nucleic acid sequences after amplification is described. We show that a DNA fragment bearing T7 RNA polymerase promoters on each extremity is able to be transcribed in two complementary RNAs, leading to a high yield direct synthesis of double-stranded RNA (dsRNA). Thus, this dsRNA can be easily detected and quantified in solution by fluorescence in the presence of propidium iodide.

Comparison of three primer sets for the detection of Mycobacterium tuberculosis in clinical samples by polymerase chain reaction.

A number of studies have underlined the interest of the polymerase chain reaction (PCR) in the detection of Mycobacterium tuberculosis in clinical samples. Among the different parameters to be carefully studied the choice of target gene and primers is essential. The amplification of nucleotidic sequences localised on three different target genes (groEL, IS6110, Pab) was examined in 196 clinical samples from patients with suspected tuberculosis or receiving antituberculous therapy.

Fast solid support detection of PCR amplified viral DNA sequences using radioiodinated or hapten labelled primers.

Oligonucleotides with novel modifications have been synthesized and incorporated into enzymatically amplified DNA sequences. They allow the fast detection of viral DNA sequences after two rounds of amplification. The hybrids formed are immobilized by affinity on coated tubes and detected by direct beta (32P) or gamma (125I) counting or by colorimetric revelation.

Histochemical detection of the messenger RNAs coding for calcitonin and calcitonin gene-related peptide in medullary thyroid carcinomas with radioactive and biotinylated oligonucleotide probes.

The present study has been undertaken to investigate the efficiency of biotinylated synthetic oligonucleotide probes in detection by in situ hybridization of the mRNAs coding for calcitonin (CT) or calcitonin gene-related peptide (CGRP) in human medullary thyroid carcinomas (MTCs). Tissue sections fixed with formaldehyde were hybridized with 45-base long oligonucleotides, specific for CT or CGRP mRNA. Recombinant DNA probe or synthetic oligonucleotides radioactively labelled with 32P or 35S were used as controls to detect by autoradiography the corresponding mRNAs in the tumour cells.

Dopamine receptor gene expression by enkephalin neurons in rat forebrain.

In situ hybridization experiments were performed with brain sections from normal, control and haloperidol-treated rats to identify and map the cells expressing the D2 dopamine receptor gene. D2 receptor mRNA was detected with radioactive or biotinylated oligonucleotide probes. D2 receptor mRNA was present in glandular cells of the pituitary intermediate lobe and in neurons of the substantia nigra, ventral tegmental area, and forebrain, especially in caudate putamen, nucleus accumbens, olfactory tubercle, and piriform cortex.

Vasopressin gene expression in the normal and Brattleboro rat: a histological analysis in semi-thin sections with biotinylated oligonucleotide probes.

We analyzed expression of the vasopressin (AVP) gene in semi-thin sections in normal and Brattleboro rats by using in situ hybridization and immunohistochemistry. AVP mRNA was detected as follows: vibratome sections of rat hypothalamus were hybridized with a biotinylated oligonucleotide probe, embedded in Araldite, and cut into semi-thin sections which were reacted with streptavidin-alkaline phosphatase and the appropriate substrate. Adjacent serial sections were treated by immunohistochemistry to detect AVP or oxytocin immunoreactivity.

Histological detection of messenger RNAs with biotinylated synthetic oligonucleotide probes.

We achieved histological detection of the messenger RNAs coding for vasopressin, calcitonin, or calcitonin gene-related peptide by using biotinylated synthetic oligonucleotides, and defined the technical parameters enabling optimal detection of these mRNAs. Oligonucleotides labeled by fixation of one biotin at their 5' end or by addition of a biotin-11-dUTP tail at their 3' end can be used to detect mRNAs, although the latter are more sensitive. Streptavidin-alkaline phosphatase revealed with nitroblue tetrazolium-bromo-chloro-indolyl phosphate as substrate makes possible detection of the biotinylated oligonucleotides.

Synthesis and spectroscopic properties of two classes of 5,6-dihydrothymidine derivatives. Action on the Ehrlich's ascites cells thymidine kinase.

Trans (+) and (-) 6-alkoxy-5-bromo-5,6 dihydrothymidine and trans (+) and (-) 6-alkoyloxy-5-bromo-5,6-dihydrothymidine compounds have been prepared. The synthesis of these substances (alkoxy : methoxy, ethoxy, butyloxy and isoamyloxy and alkoyloxy : acetoxy and bensovloxy) is described. Diastereoisomers of all products have been isolated by thin layer chromatography and their spectroscopic properties (IR, UV, NMR, mass spectrometry) studied.