OBJECTIVETo study the expression pattern of cd99l2 gene during zebrafish development, the RNA probes for whole-mount in situ hybridization were prepared in this study.

METHODSThe cd99l2 fragment obtained by RT-PCR was cloned into pGM-T Easy, then the plasmids were linearized with the restriction enzymes SacII or SalI. Using Sp6 or T(7) RNA polymerase, the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole-mount in situ hybridization.

RESULTSThe plasmid cd99l2/pGM-T was constructed. cd99l2 gene expression pattern during embryogenesis of zebrafish was examined using the antisense probe, and intense expression was detected in the central nervous system during zebrafish development.

CONCLUSIONThe antisense probe can be used for study of the spatial and temporal distribution of cd99l2 during zebrafish development using the sense probe as control.

Animals ; Central Nervous System ; embryology ; Cloning, Molecular ; Digoxigenin ; chemistry ; Gene Expression Regulation, Developmental ; Oligonucleotide Probes ; RNA Probes ; Uridine Triphosphate ; chemistry ; Zebrafish ; embryology ; genetics ; Zebrafish Proteins ; genetics

The oligonucleotide microarray, a high-throughput polymorphism detection technology, holds great promise for the characterization of complex genetic variance. To achieve greater sensitivity and specificity for it to be an effective platform technology we present results and discuss some of the factors influencing signal intensities and single-mismatch discrimination in array-based mutation/SNP detection. Probes with a series of concentrations were spotted onto the slide in order to find the optimal concentration with the identifiable satisfying signals and the stable ratios between matched and mismatched probes. It was found that under our experimental conditions, when the initial probe concentration is higher than the maximum immobilization capability of the slide (7.5 micrometer), the hybridization signal will be saturated and the ratio between matched and mismatched probes will be more stable than at a lower probe concentration. Considering the cost of probes and the systematic stability, a constant spotting concentration of 10 micrometer was selected. The stability of different types of mismatched oligo-DNA duplexes on the glass surface was also confirmed. The results show that the order of stability of mismatched oligo-DNA duplexes on a glass surface is in general agreement with previous reports conducted using liquid and polyacrylamide gel pads. This suggests that the influence of the mismatched base pair on the stability of the duplex in a solid hybridization system is similar to that in the solution hybridization environment.
Nucleic Acid Heteroduplexes/chemistry ; *Nucleic Acid Hybridization ; *Oligonucleotide Array Sequence Analysis ; Oligonucleotide Probes/chemistry ; *Polymorphism, Single Nucleotide ; Research Support, Non-U.S. Gov't

We report here a novel membrane transfer-based DNA detection method, in which alkaline phosphatase labeled gold nanoparticle (AuNP) probes were used as a means to amplify the detection signal. In this method, the capture probe P1, complimentary to the 3' end of target DNA, was immobilized on the chip. The multi-component AuNP probes were prepared by co-coating AuNPs with the detecting probe P2, complimentary to the 5' end of target DNA, and two biotin-labeled signal probes (T10 and T40) with different lengths. In the presence of target DNA, DNA hybridization led to the attachment of AuNPs on the chip surface where specific DNA sequences were located in a "sandwich" format. Alkaline phosphatase was then introduced to the surface via biotine-streptavidin interaction. By using BCIP/NBT alkaline phosphatase color development kit, a colorimetric DNA detection was achieved through membrane transfer. The signal on the membrane was then detected by the naked eye or an ordinary optical scanner. The method provided a detection of limit of 1 pmol/L for synthesized target DNA and 0.23 pmol/L for PCR products of Mycobacterium tuberculosis 16S rDNA when the ratio of probes used was 9:1:1 (T10:T40:P2). The method described here has many desirable advantages including high sensitivity, simple operation, and no need of sophisticated equipment. The method can be potentially used for reliable biosensings.
Colorimetry ; methods ; DNA Probes ; chemistry ; genetics ; DNA, Bacterial ; genetics ; Gold ; chemistry ; Humans ; Metal Nanoparticles ; chemistry ; Mycobacterium tuberculosis ; isolation & purification ; Nucleic Acid Hybridization ; methods ; Oligonucleotide Array Sequence Analysis ; methods

OBJECTIVETo detect the methylenetetrahydrofolate reductase(MTHFR) gene C677T mutation with molecular beacon technique and assess the revant applicability.

METHODSA total of 228 samples were analyzed using molecular beacons which are oligonucleotide probes to become fluorescent upon hybridization. Wild-type molecular beacon and mutant beacon were designed to detect the genotypes of MTHFR gene.

RESULTSAnalysis of the 228 samples indicated that there were three genotypes including 41 homozygous mutants, 113 heterozygous individuals and 74 wild-type individuals. Every sample was identified clearly.

CONCLUSIONThe present method, a closed-tube PCR/hybridization assay, is a simple, high-throughput and fast procedure that is fully automated for detecting gene mutation.

DNA Mutational Analysis ; methods ; Fluorescent Dyes ; chemistry ; Genotype ; Humans ; Methylenetetrahydrofolate Reductase (NADPH2) ; genetics ; Oligonucleotide Probes ; chemistry ; genetics ; Point Mutation ; Sensitivity and Specificity

OBJECTIVEStudy blood vessel injury and gene expression indicating vascular endothelial cell apoptosis induced by mannitol with and without administration of anti-oxidative vitamins.

METHODSHealthy rabbits were randomly divided into four groups. Mannitol was injected into the vein of the rabbit ear in each animal. Pre-treatment prior to mannitol injection was performed with normal saline (group B), vitamin C (group C) and vitamin E (group D). Blood vessel injury was assessed under electron and light microscopy. In a second experiment, cell culture specimen of human umbilical vein endothelial cells were treated with mannitol. Pre-treatment was done with normal saline (sample B), vitamin C (sample C) and vitamin E (sample D). Total RNA was extracted with the original single step procedure, followed by hybridisation and analysis of gene expression.

RESULTSIn the animal experiment, serious blood vessel injury was seen in group A and group B. Group D showed light injury only, and normal tissue without pathological changes was seen in group C. Of all 330 apoptosis-related genes analysed in human cell culture specimen, no significant difference was seen after pre-treatment with normal saline, compared with the gene chip without pre-treatment. On the gene chip pre-treated with vitamin C, 45 apoptosis genes were down-regulated and 34 anti-apoptosis genes were up-regulated. Pre-treatment with vitamin E resulted in the down-regulation of 3 apoptosis genes.

CONCLUSIONVitamin C can protect vascular endothelial cells from mannitol-induced injury.

Animals ; Antioxidants ; pharmacology ; Apoptosis ; Endothelial Cells ; cytology ; pathology ; Gene Expression Regulation ; Humans ; Mannitol ; chemistry ; pharmacology ; Nucleic Acid Hybridization ; Oligonucleotide Array Sequence Analysis ; Oligonucleotide Probes ; chemistry ; Oxidation-Reduction ; Rabbits ; Vitamins ; metabolism

To screen the candidate genes associated with Musca domestica antibacterial peptides using DNA microarray technique, the hybrid probes were designed from the conservative domains of the encoded area of the insect antibacterial peptide genes in GenBank with biology software Designer 2.0, and were synthesized by a chemical process, with the assistance of the automated Gen III Microarray Spotter, those oligo probes were printed on a special ready-made glass, and a cDNA microarray was constructed. The total RNA was extracted from the fat body of Musca domestic third-instar larve induced after 24 hours by Escherichia coli and Staphylococcus aureus, the strands of cDNA were labled with fluoresceine Cy3 using the method of reverse transcription PCR, after prehybridization, hybridization and washing procedure, the results of hybridization were scanned using computer system, and the data were analyzed using the software of MIDAS, fifteen valid hybridization signals were detected through two times of hybridization and scanning (the positive samples as a control were excluded). DNA microarray technique can be successfully applied screen the candidate genes associated with Musca domestica antibacterial peptides, and further provide significant evidence to discover its antibacterial peptide new genes.
Animals ; Antimicrobial Cationic Peptides ; genetics ; Base Sequence ; Gene Expression Profiling ; Genes, Insect ; Houseflies ; genetics ; growth & development ; Larva ; genetics ; growth & development ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; methods ; Oligonucleotide Probes ; chemistry

The aim of this study was to develop and validate an oligonucleotide suspension array for rapid identification of 15 bacterial species responsible for bacteremia, particularly prevalent in Chinese hospitals. The multiplexed array, based on the QIAGEN LiquiChip Workstation, included 15 oligonucleotide probes which were covalently bound to different bead sets. PCR amplicons of a variable region of the bacterial 23S rRNA genes were hybridized to the bead-bound probes. Thirty-eight strains belonging to 15 species were correctly identified on the basis of their corresponding species-specific hybridization profiles. The results show that the suspension array, in a single assay, can differentiate isolates over a wide range of strains and species, and suggest the potential utility of suspension array system to clinical laboratory diagnosis.
Bacteremia ; diagnosis ; genetics ; Bacterial Typing Techniques ; Bacteriological Techniques ; DNA Probes ; Genetic Techniques ; Listeria monocytogenes ; metabolism ; Nucleic Acid Hybridization ; Oligonucleotide Array Sequence Analysis ; Oligonucleotides ; chemistry ; RNA, Ribosomal ; chemistry ; RNA, Ribosomal, 23S ; genetics ; Stem Cells

In order to identify the differentially expressing gene of bone marrow mesenchymal stem cells (MSCs) stimulated by electromagnetic field (EMF) with osteogenesis microarray analysis, the bone marrow MSCs of SD rats were isolated and cultured in vitro. The third-passage cells were stimulated by EMFs and total RNA was extracted, purified and then used for the synthesis of cDNA and cRNA. The cRNA of stimulated group and the control group was hybridized with the rat oligo osteogenesis microarray respectively. The hybridization signals were acquired by using X-ray film after chemiluminescent detection and the data obtained were analyzed by employing the web-based completely integrated GEArray Expression Analysis Suite. RT-PCR was used to identify the target genes: Bmp1, Bmp7, Egf and Egfr. The results showed that 19 differentially expressing genes were found between the stimulated group and the control group. There were 6 up-regulated genes and 13 down-regulated genes in the stimulated group. Semi-quantitative RT-PCR confirmed that the expressions of Bmp1, Bmp7 mRNA of the stimulated group were up-regulated (P<0.05) and those of Egf, Egfr were down-regulated (P<0.05). It was suggested that the gene expression profiles of osteogenesis of the bone marrow MSCs were changed after EMF treatment. It is concluded that the genes are involved in skeletal development, bone mineral metabolism, cell growth and differentiation, cell adhesion etc.
Bone Marrow Cells/*cytology ; Cell Differentiation ; Down-Regulation ; Electromagnetic Fields ; Gene Expression Profiling ; Gene Expression Regulation ; Mesenchymal Stem Cells/*cytology ; Nucleic Acid Hybridization ; Oligonucleotide Probes/chemistry ; Osteogenesis/*genetics ; RNA, Complementary/metabolism ; Reverse Transcriptase Polymerase Chain Reaction