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PubMed2005

A multiplex quantitation method for eicosanoids and platelet-activating factor using column-switching reversed-phase liquid chromatography-tandem mass spectrometry.

Eicosanoids and platelet-activating factor (PAF) are phospholipid-derived lipid mediators produced by various tissues and cells through a cascade pathway. For a comprehensive analysis of these lipid mediators, a simultaneous quantitation method with sensitivity and reliability is necessary. This article details a development of column-switching reversed-phase liquid chromatography-tandem mass spectrometry for multiplex quantitation of eicosanoids and PAF. The adsorptive nature of lipids caused significant loss of signal in a conventional column-switching configuration. The use of an online-dilution method allowed use of 100% methanol as a sample solvent, which prevented sample adsorption to contacting surfaces. Addition of 0.2% formic acid to the sample solvent was required for the successful introduction of LTC4 to the trapping column and minimizing its carryover. The optimized method provided rapid analysis of 14 lipid mediators with a throughput of 96 samples/24 h, lower limits of quantitation of 5 pg on column, and linear calibration ranges up to 2000-5000 pg. The system was highly compatible with solid-phase-extracted samples, as methanol-eluted fractions were directly injected without reconstitution. The analysis of lipid mediator production of macrophage-like RAW264.7 cells demonstrated that the cell-based assay can be performed in a 96-well format, suitable for metabolomics analyses and/or screening strategies.

Animals↗
PubMed2005

DigiTag assay for multiplex single nucleotide polymorphism typing with high success rate.

As a consequence of Human Genome Project and single nucleotide polymorphism (SNP) discovery projects, several millions of SNPs, which include possible susceptibility SNPs for multifactorial diseases, have been revealed. Accordingly, there has been a strong drive to perform the investigation with all candidate SNPs for a certain disease without decreasing the number of analyzed SNPs. We developed DigiTag assay, which uses well-designed oligonucleotides called DNA coded numbers (DCNs) in multiplex SNP genotype analysis. During the analysis, the information of a genotype is converted to one of the DCNs in a one to one manner using oligonucleotide ligation assay (encoding). After the encoding reaction, only the DCNs regions and not the SNP specific regions are amplified using the universal primers and then SNP genotype is read out using DNA capillary arrays. DigiTag assay was found to be successful in SNP genotyping, giving a high success rate (24 of 27 SNPs) for randomly chosen SNPs. Moreover, this assay has the potential to analyze almost all kinds of the target SNPs by applying mismatch-induced probes and redesigned primer pairs at a low-cost.

DNA↗
PubMed2006

Preparation of silica encapsulated quantum dot encoded beads for multiplex assay and its properties.

Novel -COOH modified polystyrene beads were prepared by sulfonation grafting, and the surface area and pore volume are greatly improved in comparison with the swelling-treated beads. The optimization coating time is 4 h, and the corresponding -COOH content is approximately 2.1 mmol/g. The scanning electron microscope results show that the silica particles deposited on the beads and formed a silica shell that decreases the leakage of quantum dots (QDs) preferably and improves the bar code stability greatly. The anti-photobleaching of silica-coated beads was studied systemically, and the results show that the half-decay time (t1/2) of the coated beads increases to 537 s--seven times longer than that of the uncoated ones. Further DNA probe hybridization experiments indicated that the coding signal and target signal can be detected simultaneously and that the assays based on these probe-conjugated silica/QD/polystyrene beads have good specificity and sensitivity that can detect a concentration as low as 0.01 microg/ml target DNA in denatured calf thymus DNA solution, indicating that it is feasible to use this kind of bead for multiplex analysis.

DNA↗
PubMed2006

Investigation on glycosylation patterns of proteins from human liver cancer cell lines based on the multiplexed proteomics technology.

Glycosylation, a very important post-translational modification of proteins, is increasingly coming into notice. However, large-scale, throughput investigations on glycosylated proteins are few. We applied a sensitive and fast fluorescence-based multiplexed proteomics (MP) technology which included two-dimensional gel electrophoresis (2-DE) followed by the fluorescence staining of glycoprotein and mass spectrometry identification for the purpose of constructing glycoprotein databases of the typical human hepatocellular carcinoma cell lines including Hep3B cell line without metastasis and MHCC97H with highly metastatic potential as well as the control non-tumor Chang liver cell. 74+/-2 (n=3), 78+/-3 (n=3) and 72+/-5 (n=3) glycoprotein spots were detected on 2-DE gels from Chang liver, Hep3B and MHCC97H cell sample using this MP technique, respectively. In all, 80 glycoproteins from three cell lines were successfully identified via peptide mass profiling using MALDI-TOF-MS/MS and the identified glycoproteins were annotated to our databases. In addition, we also found the glycosylation pattern differences among these three cell lines. The protein glycosylation alteration would be have great significance for the diagnosis of HCC and prediction of its metastasis. This study described the construction of glycosylation patterns of proteins and glycoproteome databases of human liver cells by the novel technological platform. The glycoproteome databases also provide essential basis for following study.

Animals↗
PubMed2005

Multiplex real-time PCR for detection of deletions and duplications in dystrophin gene.

Genetic testing of Duchenne and Becker muscular dystrophies (DMD/BMD) is a difficult task due to the occurrence of deletions or duplications within dystrophin (DMD) gene that requires dose sensitive tests. We developed three multiplex quantitative real-time PCR assays for dystrophin exon 5, 45, and 51 within two major hotspots of deletion/duplication. Each exon was co-amplified with a reference X-linked gene and the copy number of the target fragment was calculated by comparative threshold cycle method (delta deltaC(t)). We compared the performance of this method with previously described end-point PCR fluorescent analysis (EPFA) by studying 24 subjects carrying DMD deletions or duplications. We showed that Q-PCR is an accurate and sensitive technique for the identification of deletions and duplications in DMD/BMD. Q-PCR is a valuable tool for independent confirmation of EPFA screening, particularly when deletions/duplications of single exons occur or for rapid identification of known mutations in at risk carriers.

DNA Primers↗
PubMed2006

Multiplexing RT-PCR for the detection of multiple miRNA species in small samples.

MicroRNAs are short (approximately 22 nucleotides), non-coding RNAs that play critical roles in gene regulation and may be used as rapid precise diagnostic indicators of early stages of cancer. The small size of these RNAs makes detection of multiple microRNA species in very small samples problematic. Here we investigate the parameters associated with multiplexing RT-PCR to obtain relative abundance profiles of multiple microRNAs in small sample sizes down to the amount of RNA found in a single cell.

Humans↗
PubMed2006

Multiplex primer extension reaction screening and oxidative challenge of glucose-6-phosphate dehydrogenase mutants in hemizygous and heterozygous subjects.

The primary objective of our study was to provide a simple and reliable assay for identifying the majority of G6PD genetic variants in the Chinese population. We optimized the multiplex primer extension reaction (MPER) assay for simultaneous screening of 14-point mutations in 98 G6PD-deficient subjects. Our data demonstrated that this method is precise, cost-effective and has successfully identified mutations in 97 out of 98 subjects, including all heterozygous mutants. We also detected a relatively high incidence (12.3%) of c.871G > A, and all of them harbored the silent mutation c.1311C > T. Apart from the screening program, the pharmacogenetic relationship between G6PD level and residual reduced glutathione (GSH) level was studied upon oxidative challenge by alpha-naphthol. The GSH levels were correlated with their status of G6PD deficiency, but no significant difference was observed between individual G6PD-deficient groups. Our data demonstrated the potentials of the MPER assay for characterization of G6PD deficiency and other genetic diseases.

China↗
PubMed2004

Oligonucleotide ligation assay-based DNA chip for multiplex detection of single nucleotide polymorphism.

An oligonucleotide ligation assay-based DNA chip has been developed to detect single nucleotide polymorphism. Synthesized nonamers, complementary to the flanking sequences of the mutation sites in target DNA, were immobilized onto glass slides through disulfide bonds on their 5' terminus. Allele-specific pentamers annealed adjacent to the nonamers on the complementary target DNA, containing 5'-phosphate groups and biotin labeled 3'-ends, were mixed with the target DNA in tube. Ligation reactions between nonamers and pentamers were carried out on chips in the presence of T4 DNA ligase. Ligation products were directly visualized on chips through enzyme-linked assay. The effect of G:T mismatch at different positions of pentamers on the ligation were evaluated. The results showed that any mismatch between pentamer and the target DNA could lead to the decrease of ligation, which can be detected easily. The established approach was further used for multiplex detection of mutations in rpoB gene of rifampin-resistant Mycobacterium tuberculosis clinical isolates.

DNA↗
PubMed2004

Identification and characterization of Bacillus anthracis by multiplex PCR on DNA chip.

Bacillus anthracis can be identified by detecting virulence factor genes located on two plasmids, pXO1 and pXO2. Combining multiplex PCR with arrayed anchored primer PCR and biotin-avidin alkaline phosphatase indicator system, we developed a qualitative DNA chip method for characterization of B. anthracis, and simultaneous confirmation of the species identity independent of plasmid contents. The assay amplifies pag gene (in pXO1), cap gene (in pXO2) and Ba813 gene (a B. anthracis specific chromosomal marker), and the results were indicated by an easy-to-read profile based on the color reaction of alkaline phosphatase. About 1 pg of specific DNA fragments on the chip wells could be detected after PCR. With the proposed method, the avirulent (pXO1+/2-, pXO1-/2+ and pXO1-/2-) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria were unambiguously identified, while the genera other than Bacillus gave no positive signal.

Alkaline Phosphatase↗
PubMed2005

Arthrogryposis multiplex congenita with callosal agenesis and dentato-olivary dysplasia.

We report the autopsy case of a boy with arthrogryposis multiplex congenita, associated with callosal agenesis and dentato-olivary dysplasia. The patient manifested with dysmorphic facial features and suffered from intractable epilepsy during the neonatal period. These sets of complications suggest that a common molecular mechanism may be involved in the development of corpus callosum and the folding of the dentate and inferior olivary nuclei. Deep brain structures, including the brainstem and the cerebellum, may be involved in the pathophysiology of symptomatic generalized epilepsy. The differential diagnoses for the clinical and pathological characteristics of this patient are discussed.

Agenesis of Corpus Callosum↗
PubMed2005

Molecular cytogenetic characteristics of the human hepatocellular carcinoma cell line HCCLM3 with high metastatic potential: comparative genomic hybridization and multiplex fluorescence in situ hybridization.

The HCCLM3 cell line was established at the authors' institute from the lung metastatic lesions of BALB/c nude mice bearing human hepatocellular carcinoma (HCC) from the metastatic HCC cell line MHCC97-H. It has been shown to have a high potential for lung metastases and extensive metastases when the cells are inoculated subcutaneously or orthotopically in athymic nude mice. In the present study, the molecular cytogenetic characteristics of this cell line were evaluated with conventional G-banding, comparative genomic hybridization, and multiplex fluorescence in situ hybridization. A hyperdiploid karyotype of 53-58 chromosomes with 10 marker chromosomes was identified. The chromosomal aberrations such as i(X)(q10), der(Y)t(Y;18)(q12;p11), der(3)t(3;20) (p25;q13), der(4)t(4;8)(q31;q22)5, der(9)t(9;13)(p21;q22), der(14)t(14;22)(p13;q13), and der(15) t(15;21)(q11;q22) were described for the first time in human HCC cells. The analysis of this cell line through a combination of molecular cytogenetic techniques provides information on the possible molecular mechanisms involved in the metastatic process of HCC.

Animals↗
PubMed2005

Role of multiplex FISH in identifying chromosome involvement in myelodysplastic syndromes and acute myeloid leukemias with complex karyotypes: a report on 28 cases.

Chromosomal abnormalities are found by conventional cytogenetic (CC) analysis in about 50% of myelodysplastic syndromes (MDS) and 70% of acute myeloid leukemias (AML). When cytogenetic abnormalities are complex, multiplex fluorescence in situ hybridization (M-FISH) can help clarify complex chromosomal abnormalities and identify rearrangements with prognostic value or cryptic translocations, which could be preliminary steps in identifying new genes. We studied by M-FISH 28 cases of MDS and AML with complex chromosomal abnormalities, 10 of them were therapy-related. M-FISH allowed the characterization of unidentified chromosomal material in 26 cases (93%). One or several unbalanced rearrangements were observed in 27 cases (96%), generally interpreted as deletions or additional material by CC. Among those translocations, 4 involved 3 chromosomes. Eighteen cryptic translocations undetected by CC were found in 13 cases. By FISH analysis using locus specific probes, TP53 deletion, additional copies of MLL, and additional copies or deletions of RUNX1/AML1 were observed in 16, 4, and 3 cases, respectively. Thus, M-FISH is an important tool to characterize complex chromosomal abnormalities which identified unbalanced and cryptic translocations in 96% and 46% of the cases studied, respectively. Complementary FISH helped us identify involvement of TP53, MLL, and RUNX1/AML1 genes in 82% of cases, confirming their probable role in leukemogenesis.

Acute Disease↗
PubMed2005

Characterization of 3p, 5p, and 3q in two nasopharyngeal carcinoma cell lines, using region-specific multiplex fluorescence in situ hybridization probes.

Amplification of chromosome arms 3q and 5p and deletion of 3p were frequently detected in nasopharyngeal carcinoma (NPC) with comparative genomic hybridization and loss of heterozygosity studies. To identify the minimal amplified or deleted regions in these arms, structural aberrations in chromosome arms 3p, 3q, and 5p in two NPC cell lines, CNE1 and SUNE1, were studied with multiplex-color FISH (M-FISH) and chromosome region-specific probes (CRP). All CRPs, which were generated from microdissected DNA, were specific and strong in intensity, and sensitive enough to detect chromosome aberrations including translocations, deletions, and amplifications of target regions. In these two NPC cell lines, minimal regions of deletion and amplification were found at 3p12 and 3q26 approximately q27, respectively. On 5p, most of the regions were amplified as intact copies. Interregion translocations of these three arms were also observed. The amplification on 3q26 approximately q27 provided useful hints for further screening the minimal amplification at RP11-115J24 (3q26.2), containing candidate oncogene eIF-5A2. M-FISH with CRPs is thus not only useful in revealing a comprehensive picture of structural aberrations in target chromosomes, but also in narrowing down the minimal region for screening cancer-related genes.

Cell Line, Tumor↗
PubMed2006

Cytogenetic studies of a series of 43 consecutive secondary myelodysplastic syndromes/acute myeloid leukemias: conventional cytogenetics, FISH, and multiplex FISH.

We report a series of 43 consecutive therapy-related myelodysplastic syndromes (t-MDS) or acute myeloid leukemias (t-AML) observed for 6 years. This series consisted of 26 women and 17 men, ages ranging from 9 to 85 years. These cases were classified into three groups according to the primary diagnosis. Conventional cytogenetic and fluorescent in situ hybridization (FISH)/ multiplex FISH (M-FISH) methods were used to analyze cytogenetic characteristics of secondary MDS/AML. The features of chromosomal abnormalities were linked to the nature of the therapy and protocols used. A considerable proportion of recurrent balanced translocations characterized t-AML secondary to therapy. FISH techniques showed that conventional cytogenetics often underestimated associated translocations; some deletions were in fact derivative chromosomes associated with deletions. After treatment for lymphomas and chronic myeloproliferative diseases, there were more complex unbalanced abnormalities than the control group. Compared to other series, recurrent translocations appeared to be more numerous (25%), probably reflecting an evolution of therapeutic modalities.

Adolescent↗
PubMed2006

Characterization of rearrangements involving 4q, 13q and 16q in hepatocellular carcinoma cell lines using region-specific multiplex-FISH probes.

Deletions in 4q, 13q and 16q were frequently detected in hepatocellular carcinoma (HCC) by comparative genomic hybridization (CGH) studies. However, detailed chromosome structural aberrations are not fully explored. Using CGH combined with multiplex-color FISH (M-FISH) with chromosome region-specific probes (CRPs), chromosome structural aberrations in 4q, 13q and 16q in six HCC cell lines were studied. All CRPs, which were generated from microdissected DNA, were specific, strong in intensity and sensitive enough to detect chromosome structural aberrations including translocation and deletion. FISH with BAC probes was used to further characterize translocation breakpoints and deletions. A breakpoint at 16q22 was localized at a BAC clone (RP11-341K23) and another breakpoint at 4q28 was localized within a 620 kb-region. A minimal deleted region at 13q21 was found between BAC clones RP11-240M20 and RP11-435P18. This study demonstrated that the combination of CGH, M-FISH and BAC-FISH is a very useful tool to detect and characterize translocation breakpoint.

Carcinoma, Hepatocellular↗
PubMed2004

A multiplexed allele-specific polymerase chain reaction assay for the detection of common thiopurine S-methyltransferase (TPMT) mutations.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs that are commonly used to treat a wide range of conditions. It is now well established that interpatient variation in sensitivity to these drugs is due to point mutations in the TPMT gene. The mutant alleles TPMT*2 (238G>C), TPMT*3A (460G>A, 719A>G), TPMT*3B (460G>A), and TPMT*3C (719A>G) account for 80-95% of TPMT deficiency observed in Caucasian populations. In this paper, we describe a novel, multiplex, allele-specific polymerase chain reaction (PCR) method that detects the 238G>C, 460G>A, and 719A>G mutations, allowing for the simultaneous identification of TPMT*2 and TPMT*3 alleles. The assay is internally controlled, robust, and does not require the use of restriction endonucleases. Therefore, the assay is not prone to erroneous readings due to incomplete restriction digestion, as documented for existing PCR restriction fragment length polymorphism (RFLP) assays of TPMT.

Alleles↗
PubMed2004

A simple multiplex PCR method for the concurrent detection of three CYP2C8 variants.

BACKGROUND: Cytochrome P450 (CYP) 2C8 is a principle enzyme responsible for the metabolism of many clinically important drugs as well as endogenous compounds such as arachidonic acid. The enzyme is genetically polymorphic but a simple method is not available to study its genetic polymorphism. We developed and optimized a variant-specific PCR techniques to detect CYP2C8*2, CYP2C8*3 and CYP2C8*4. METHOD: Genomic DNA was extracted from blood using standard extraction methods. A two-step PCR method was developed to detect simultaneously three CYP2C8 variants. In the first PCR (PCR1), specific regions from exons 3, 5 and 8 of the CYP2C8 gene were amplified. The products were used as templates in parallel alleles-specific PCR (PCR2). This method was tested against DNA samples obtained from 57 healthy Malaysian volunteers. RESULT: The bands of interest were successfully amplified. This method showed specific and reproducible results when tested on healthy volunteers. DNA sequencing further confirmed genotype results obtained from current method. CONCLUSION: We have successfully developed and optimized a multiplex PCR method suitable for use in population studies of CYP2C8 polymorphism.

Alleles↗