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At least 145 records · Page 8Linked to original sources

PubMed2005

Small interfering RNA and gene expression analysis using a multiplex branched DNA assay without RNA purification.

The authors have developed a novel multiplex detection system that quantitatively measures the expression level of 11 messenger RNAs (mRNAs) directly from cell lysates or tissue homogenates without RNA purification. The system incorporates branched DNA (bDNA) technology from Bayer and a multiplex bead array platform from Luminex. In this study, a 21-nt synthetic small interfering RNA (siRNA; specifically designed to knockdown interleukin-8 [IL-8] expression) was delivered into HeLa cells. Using the multiplex bDNA assay, gene expression levels were measured simultaneously from cell lysates for 11 genes. After treating the HeLa cells for 20 h with phorbol myristate acetate (PMA), IL-8 mRNA levels were induced by almost 50-fold; transfection with 30 nM IL-8-specific siRNA reduced the PMA-induced IL-8 mRNA by 80%. In addition, PMA induced mRNA expression in IL-1alpha (3-fold) and IL-6 (4-fold); however, the IL-8 siRNA did not affect the expression of either of these 2 cytokine genes, indicating that the siRNA was selective for IL-8 mRNA expression. Three housekeeping genes' expression levels were measured under all conditions tested. The multiplex bDNA assay provides a powerful tool for quantitative multiplex gene expression analysis directly from cell lysates, which could be extremely valuable for conservation of rare or difficult-to-obtain samples.

Cytokines↗

PubMed2005

Multiplexed G-protein-coupled receptor Ca2+ flux assays for high-throughput screening.

An early drug discovery approach focusing on gene families can benefit from strategies that exploit common signaling mechanisms to more effectively identify and characterize novel chemical lead structures. Multiplexing, defined as the screening of multiple targets within the same experiment, is an example of this strategy. Here, the authors describe a technique that allows multiplexing of a common assay type used to study G-protein-coupled receptors: changes in intracellular Ca2+ levels as measured by Molecular Device's fluorometric imaging plate reader (FLIPR). The multiplexed FLIPR assays showed the expected pharmacological properties of single assays, with good reproducibility and Z* factors. The authors used them to screen large compound libraries in 2 multiplexed assay designs. The 1st used a single-cell line expressing 2 different receptors and the 2nd a mixture of 2 cell lines of the same type each expressing distinct receptors. Screening using these multiplexed assays produced significant savings in reagents, time, and human resources and allowed the authors to quickly identify specific and selective hits.

Biological Assay↗

PubMed2006

Detection of Norovirus genogroup I and II by multiplex real-time RT- PCR using a 3'-minor groove binder-DNA probe.

BACKGROUND: Due to an increasing number of norovirus infections in the last years rapid, specific, and sensitive diagnostic tools are needed. Reverse transcriptase-polymerase chain reactions (RT-PCR) have become the methods of choice. To minimize the working time and the risk of carryover contamination during the multi-step procedure of PCR the multiplex real-time RT-PCR for the simultaneous detection of genogroup I (GI) and II (GII) offers advantages for the handling of large amounts of clinical specimens. METHODS: We have developed and evaluated a multiplex one-tube RT-PCR using a combination of optimized GI and GII specific primers located in the junction between ORF1 and ORF2 of the norovirus genome. For the detection of GI samples, a 3'-minor groove binder-DNA probe (GI-MGB-probe) were designed and used for the multiplex real-time PCR. RESULTS: Comparable results to those of our in-house nested PCR and monoplex real-time-PCR were only obtained using the GI specific MGB-probe. The MGB-probe forms extremely stable duplexes with single-stranded DNA targets, which enabled us to design a shorter probe (length 15 nucleotides) hybridizing to a more conserved part of the GI sequences. 97% of 100 previously norovirus positive specimens (tested by nested PCR and/or monoplex real-time PCR) were detected by the multiplex real-time PCR. A broad dynamic range from 2 x 10(1) to 2 x 10(7) genomic equivalents per assay using plasmid DNA standards for GI and GII were obtained and viral loads between 2.5 x 10(2) and 2 x 10(12) copies per ml stool suspension were detected. CONCLUSION: The one-tube multiplex RT real-time PCR using a minor groove binder-DNA probe for GI is a fast, specific, sensitive and cost-effective tool for the detection of norovirus infections in both mass outbreaks and sporadic cases and may have also applications in food and environmental testing.

Animals↗

PubMed2000

Detection of thermophilic Campylobacter from sparrows by multiplex PCR: the role of sparrows as a source of contamination of broilers with Campylobacter.

The best combination of primers and the annealing temperature of multiplex PCR for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari were examined. The multiplex PCR was able to detect type strains of the three species. All results of identification of wild strains (30 strains of C. jejuni, 20 strains of C. coli, and 4 strains of C. lari) by the multiplex PCR coincided with those of the conventional biochemical identification tests, suggesting that the multiplex PCR can simultaneously differentiate C. jejuni, C. coli, and C. lari from wild strains of campylobacters easily and rapidly. Campylobacters were detected from sparrow feces by the multiplex PCR and antimicrobial sensitivities of the strains were determined to discuss the role of sparrows in contamination of broilers with C. jejuni. Three out of 13 strains of C. jejuni isolated from sparrow feces showed quinolone resistance. From the frequent use of quinolones for treatment of industrial animals like chickens, pigs, and cows, the three strains of quinolone-resistant C. jejuni in sparrows must have been originated from those industrial animals. Sparrows that have quinolone-resistant C. jejuni were considered to have contacted with industrial animals or thier feed. It may be presumed, on the contrary, that C. jejuni in sparrows could be a potential source of contamination of broilers.

Animals↗

PubMed2004

Remote multiplexing of holograms with random patterns from multimode fiber bundles.

We propose the remote multiplexing of holograms with random patterns from a multimode fiber bundle used as the reference beams. The random pattern reference beam is characterized by the superposition and concatenation of propagation modes of multimode fiber and free space. For angle, shift, and wavelength remote multiplexing we compare two methods of laser coupling to the fiber bundle, i.e., direct coupling and lens coupling. A theoretical discussion that uses mode orthogonality is provided to describe multiplexing characteristics, and the theory is verified by experimental results. These remote-multiplexing methods can be applied to general multimode waveguide arrays for construction of compact and integrated optical systems in which multiplexing can be controlled remotely.

Journal Article↗

PubMed2006

Demodulation algorithm for spatial-frequency-division-multiplexed fiber-optic Fizeau strain sensor networks.

A demodulation algorithm for spatial-frequency-division-multiplexed fiber-optic Fizeau strain sensor networks with a large number of sensors is proposed to effectively reduce the cross talk between any two adjacent sensors and hence substantially enhance the multiplexing capability of the network, which is based on the Pisarenko algorithm. The cross talk between two fiber-optic Fizeau sensors is investigated experimentally. The experimental results show that a strain accuracy of better than +/-10 micro permittivity can be achieved even when the cavity length difference is approximately 100 microm. It was demonstrated that the multiplexing capability of the spatial-frequency-division-multiplexed fiber-optic Fizeau sensor network can be approximately 5 times greater than that of the conventional fast-Fourier-transform algorithm, and this can lead to achievement of a sensing network with a multiplexing capability of as many as 1000 Fizeau sensors.

Journal Article↗

PubMed2006

Virus-coated layer-by-layer colloids as a multiplex suspension array for the detection and quantification of virus-specific antibodies.

BACKGROUND: Suspension array technology has surpassed ELISA for automated, simultaneous detection and quantification of soluble biomarkers such as virus-specific antibodies. We describe assays in which antigens are attached to a lipid bilayer surrounding color-coded particles. METHODS: We used layer-by-layer technology to establish a multiplex suspension array with distinguishable microbeads coated with authentic viral surfaces to catch and quantify virus-specific antibodies in a flow cytometric analysis. Antigenic surfaces were generated by chimeric and wild-type baculoviruses plus 2 different influenza A virus subtypes fused to a lipid bilayer surrounding distinctly colored particles. Specificity of binding of chosen antibodies and sera was detected by immunofluorescence. Results of multiplex analysis were compared with results of ELISA. RESULTS: Titrations of virus-specific antibodies in the multiplex suspension array demonstrated specific binding to the viral surface proteins. The multiplex suspension array gave positive results for up to log 5-diluted primary antibodies with an approximately 5- to 10-fold reduced dynamic range compared with the respective ELISA. CONCLUSIONS: The bead-based multiplex suspension array is customizable and easy to establish. By displaying native influenza A virus surfaces and recombinant HIV-1 epitopes, the new assay provides a tool for the detection of major viral infections in humans.

Antibodies, Viral↗

PubMed1997

Multiplex PCR: critical parameters and step-by-step protocol.

By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.

Buffers↗

PubMed2003

Steatocystoma multiplex: mammographic and sonographic manifestations.

OBJECTIVE: This study was performed to identify the typical imaging features of steatocystoma multiplex on mammography and sonography. CONCLUSION: Steatocystoma multiplex is manifest by multiple clinically palpable skin-colored or yellow nodules scattered on the axillae and the anterior chest wall. Steatocystoma multiplex is included in the differential diagnoses of lipoma, fat necrosis, galactocele, epidermal cyst, and so on, so radiologists need to be aware of its distinguishing features. The findings of a well-circumscribed round fat-density nodule on mammography or an intradermal hypoechoic nodule on sonography, combined with a family history of steatocystoma multiplex (40%) and an extensive bilateral spatial distribution of nodules, confirm the diagnosis of steatocystoma multiplex.

Adult↗

PubMed2004

Simultaneous detection of HBV and HCV by multiplex PCR normalization.

AIM: To design and establish a method of multiplex PCR normalization for simultaneously detecting HBV and HCV. METHODS: Two pairs of primers with a 20 bp joint sequence were used to amplify the target genes of HBV and HCV by two rounds of amplification. After the two rounds of amplification all the products had the joint sequence. Then the joint sequence was used as primers to finish the last amplification. Finally multiplex PCR was normalized to a single PCR system to eliminate multiplex factor interference. Four kinds of nucleic acid extraction methods were compared and screened. A multiplex PCR normalization method was established and optimized by orthogonal design of 6 key factors. Then twenty serum samples were detected to evaluate the validity and authenticity of this method. RESULTS: The sensitivity, specificity, diagnostic index and efficiency were 83.3%, 70%, 153.3% and 72.2%, respectively for both HBsAg and anti-HCV positive patients, and were 78.6%, 80%, 258.6% and 79.2%, respectively for HBsAg positive patients, and were 75%, 90%, 165% and 83.3%, respectively for anti-HCV positive patients. CONCLUSION: The multiplex PCR normalization method shows a broad prospect in simultaneous amplification of multiple genes of different sources. It is practical, correct and authentic, and can be used to prevent and control HBV and HCV.

DNA↗

PubMed1999

TWGDAM validation of a nine-locus and a four-locus fluorescent STR multiplex system.

The Gene Print PowerPlex 1.1/Amelogenin and FFFL Fluorescent STR Systems have been validated following the recommendations presented by the Technical Working Group on DNA Analysis Methods (TWGDAM). The PowerPlex 1.1/Amelogenin System supports simultaneous amplification of eight short tandem repeat loci and the Amelogenin gender identification marker. The loci D16S539, D7S820, D13S317, and D5S818 are labeled with fluorescein (FL) while the loci CSF1PO, TP0X, TH01, vWA and Amelogenin are labeled with carboxy-tetramethylrhodamine (TMR). The FFFL Multiplex System is composed of the loci F13A01, FESFPS, F13B, and LPL, each labeled with fluorescein. We have observed no overlap of alleles across loci labeled with an individual fluorescent dye. Samples of each system were amplified and labeled in a single reaction, separated by electrophoresis through a denaturing polyacrylamide gel, and amplified alleles detected using a Hitachi FMBIO Fluorescent Scanner. Alterations from the standard amplification protocols in cycle number and annealing temperature generally produced excellent results. In experiments testing sensitivity as little as 0.2 ng of DNA template could be detected. As expected, different body fluids from the same individuals generated identical DNA profile results. Template DNA derived from blood-strains deposited on a variety of matrix supports displayed robust amplification except for material derived from deposits on wood and Japanese orchid leaves. Mixtures of DNA templates could be interpreted with the minor component present in as little as ten percent of the total sample. Monoplex and multiplex amplifications produced identical amplified allele patterns, indicating that STR multiplex systems save template and increase efficiency in the amplification procedure without loss of quality. Analyses of genotype frequencies in African-American, Caucasian-American and Hispanic-American populations using all twelve loci were used to determine matching probabilities smaller than 1 in 1.14 x 10(8) and 1 in 2658 for the PowerPlex 1.1 and the FFFL Multiplex Systems, respectively. The matching probability achieved with the two systems combined is smaller than 1 in 3.03 x 10(11). The independence of alleles within loci was generally demonstrated by applying the exact test to demonstrate Hardy-Weinberg Equilibrium. All of the studies performed indicate that the PowerPlex 1.1/Amelogenin and FFFL Multiplex Systems are powerful, robust, and reliable investigative tools that can be used in the analysis of forensic samples.

DNA Fingerprinting↗

PubMed2000

Detection and typing of HSV-1, HSV-2, and VZV by a multiplex polymerase chain reaction.

The development of a multiplex polymerase chain reaction method for the rapid and accurate detection and typing of HSV-1, HSV-2, and VZV from clinical specimens is described. A sensitive multiplex polymerase chain reaction was achieved by optimization of parameters such as the primers, magnesium, and dNTPs concentrations. False-negative results that sometimes arise due to inhibitors of DNA amplification or failure of DNA extraction procedure used may be avoided by assaying each specimen with alpha-tubulin primers. Multiplex PCR amplified viral sequences from all 55 specimens obtained from patients with clinical evidence of HSV or VZV infection indicated 100% sensitivity. From 55 patients who were investigated by multiplex PCR, HSV-1 was detected in 28, HSV-2 in 20, and VZV in 7 specimens. The reported results indicate that the present multiplex PCR assay has a potential application in clinical diagnosis when a rapid and accurate detection and typing of involved viruses HSV-1, HSV-2, or VZV is needed.

Adult↗

PubMedDate not supplied

A multiplex reverse transcription-polymerase chain reaction assay for Newcastle disease virus and avian pneumovirus (Colorado strain).

Newcastle disease virus (NDV) and avian pneumovirus (APV) cause Newcastle disease and rhinotracheitis respectively, in turkeys. Both of these viruses infect the respiratory system. A one-tube, multiplex, reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of both NDV and Colorado strain of APV (APV-Col) was developed and evaluated. The primers, specific for each virus, were designed from the matrix protein gene of APV-Col and the fusion protein gene of NDV to amplify products of 631 and 309 nucleotides, respectively. The multiplex RT-PCR assay, for detecting both viruses simultaneously, was compared with the single-virus RT-PCR assays for its sensitivity and specificity. The specific primers amplified products of predicted size from each virus in the multiplex as well as the single-virus RT-PCR assays. The multiplex RT-PCR assay was determined to be equivalent to the single-virus RT-PCR assays for detecting both NDV and APV-Col. This multiplex RT-PCR assay proved to be a sensitive method for the simultaneous and rapid detection of NDV and APV-Col. This assay has the potential for clinical diagnostic applications.

Animals↗

PubMed2002

Simultaneous detection of three mosquito-borne encephalitis viruses (eastern equine, La Crosse, and St. Louis) with a single-tube multiplex reverse transcriptase polymerase chain reaction assay.

Three mosquito-borne human encephalitis viruses (eastern equine encephalitis virus [EEE], St. Louis encephalitis virus [SLE], and La Crosse encephalitis virus [LAC]) are sympatric in the southeastern United States. However, little is known concerning the temporal and spatial pattern of the distribution of these viruses in this area. As part of surveillance activities to detect the transmission of these 3 viruses in the Tennessee Valley area, we developed a single-tube multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay capable of detecting these 3 mosquito-borne viruses in a single reaction. Three viruses were differentiated by size of amplified products. Sensitivities of the multiplex RT-PCR assay for SLE, EEE, and LAC were 1-3 log median tissue culture infective doses per pool, roughly comparable to the reported sensitivity of PCR detection assays for the individual viruses, and 1 log more sensitive than antigen-capture assays for SLE and EEE. The sensitivity of the multiplex PCR was not changed significantly when carried out in the presence of extracts prepared from 50 uninfected mosquitoes. The cost of the assay is estimated at $2.98 per test, similar to the cost of other RT-PCR-based assays for viruses. However, adaptation of the RT-PCR to a multiplex format adds less than $0.01 to the per-unit cost of an RT-PCR assay targeting a single virus species. Analysis of these data suggests that the single-tube multiplex RT-PCR assay represents a sensitive, specific, cost-effective, and rapid method for monitoring activities of the 3 endemic mosquito-borne human encephalitis viruses in mosquito populations in the southeastern United States.

Animals↗

PubMed2002

Identification of Salmonella enteritidis isolates by polymerase chain reaction and multiplex polymerase chain reaction.

The polymerase chain reaction and the multiplex polymerase chain reaction were developed for detection of Salmonella and for identification of the serotype enteritidis. Three sets of primers were selected from different genomic sequences amplifying a 429 bp fragment specific for the genus Salmonella within a randomly cloned sequence, including a 250 bp fragment within the spv gene, and a 310 bp fragment within the sefA gene specific for Salmonella enteritidis. The polymerase chain reaction and the multiplex polymerase chain reaction were used for detecting S. enteritidis isolated from stool samples during outbreaks of foodborne gastroenteritis between 1992 and 1998 in Taiwan. The sefA gene was detected in all 27 strains of S. enteritidis by this polymerase chain reaction method. Multiplex polymerase chain reaction could detect 3 genes in all strains, but could not detect the spv gene in 2 strains. The sensitivity of the polymerase chain reaction and the multiplex polymerase chain reaction were 10(4) and 10(5) cells/mL, respectively. In double polymerase chain reaction, the sensitivity increased to 100 cells/mL. These data indicate that the specificity and sensitivity of the polymerase chain reaction and the multiplex polymerase chain reaction make them potentially valuable tools for diagnosis of S. enteritidis infection and that they may be used for the identification of S. enteritidis responsible for sporadic enteritis cases.

Animals↗

PubMed2002

Multiplex gene expression analysis for high-throughput drug discovery: screening and analysis of compounds affecting genes overexpressed in cancer cells.

Drug discovery strategies are needed that can rapidly exploit multiple therapeutic targets associated with the complex gene expression changes that characterize a polygenic disease such as cancer. We report a new cell-based high-throughput technology for screening chemical libraries against several potential cancer target genes in parallel. Multiplex gene expression (MGE) analysis provides direct and quantitative measurement of multiple endogenous mRNAs using a multiplexed detection system coupled to reverse transcription-PCR. A multiplex assay for six genes overexpressed in cancer cells was used to screen 9000 chemicals and known drugs in the human prostate cancer cell line PC-3. Active compounds that modulated gene expression levels were identified, and IC50 values were determined for compounds that bind DNA, cell surface receptors, and components of intracellular signaling pathways. A class of steroids related to the cardiac glycosides was identified that potently inhibited the plasma membrane Na(+)K(+)-ATPase resulting in the inhibition of four of the prostate target genes including transcription factors Hoxb-13, hPSE/PDEF, hepatocyte nuclear factor-3alpha, and the inhibitor of apoptosis, survivin. Representative compounds selectively induced apoptosis in PC-3 cells compared with the nonmetastatic cell line BPH-1. The multiplex assay distinguished potencies among structural variants, enabling structure-activity analysis suitable for chemical optimization studies. A second multiplex assay for five toxicological markers, Hsp70, Gadd153, Gadd45, O6-methylguanine-DNA methyltransferase, and cyclophilin, detected compounds that caused DNA damage and cellular stress and was a more sensitive and specific indicator of potential toxicity than measurement of cell viability. MGE analysis facilitates rapid drug screening and compound optimization, the simultaneous measurement of toxicological end points, and gene function analysis.

Actins↗

PubMed2003

A multiplex reverse transcriptase-polymerase chain reaction strategy for the diagnostic molecular screening of chimeric genes: a clinical evaluation on 170 patients with acute lymphoblastic leukemia.

BACKGROUND AND OBJECTIVES: In the last few years molecular methods have allowed the identification of leukemia-associated genetic lesions, which may represent the most accurate predictors of clinical outcome. These considerations strengthen the need for rapid identification of the abnormalities. Our aim was to demonstrate whether a modified multiplex reverse transcription polymerase chain reaction (RT-PCR) system might be successfully used to screen a large number of patients with acute lymphoblastic leukemia (ALL). DESIGN AND METHODS: In this study we adapted the multiplex RT-PCR assay, previously described by Pallisgaard et al., to detect all the most frequent genetic lesions with their characteristic splicing variants occurring in acute lymphoblastic leukemia, such as the MLL/AF4, MLL/ENL, BCR/ABL p190 (e1a2) and p210 (b2a2,b3a2) isoforms, E2A/PBX1, TEL/AML1, SIL/TAL1 and the novel NUP98/RAP1GDS1 transcript, recently described in a T-ALL leukemic subtype. RESULTS: We used the multiplex RT-PCR assay to screen 170 ALL patients (70 children and 100 adults). PCR positivity was detected in 67 (39%) of the 170 ALL patients studied. The comparison between cytogenetic and molecular analyses showed complete correspondence between the two assays in all patients with an evaluable karyotype. Finally, the observed incidence of genetic lesions in our ALL patients was similar to the frequency usually reported both in children and in adults with ALL. INTERPRETATION AND CONCLUSIONS: These results show that, compared to single RT-PCR reactions, our multiplex RT-PCR system allows rapid, specific, simultaneous as well as a less expensive, laborious and time-consuming detection of the most frequent fusion transcripts in ALL patients. Therefore, it might be recommended for rapid diagnostic molecular screening of large numbers of patients, such as those enrolled in multicenter, co-operative studies. Furthermore, we have shown that multiplex RT-PCR is an open system that can easily be adapted to detect new leukemic genes.

Adult↗

PubMed2004

[A combined assay of multiplex RT-PCR and karyotypic analysis in childhood acute lymphoblastic leukemia].

OBJECTIVE: To study the value of combination assay of multiplex RT-PCR and karyotypic analysis in the diagnosis and classification of childhood acute lymphoblastic leukemia (ALL). METHODS: Fifty cases of childhood ALL patients were studied by multiplex RT-PCR in combination with R or G banding karyotype analysis. RESULTS: Of the 50 childhood ALL patients, 18 (36.0%) carried 11 types of fusion genes including E2A/PBX1, TEL/AML1, TLS/ERG, MLL/AF4, MLL/AF9, MLL/AF10, MLL/AFX, MLL/AF6, MLL/ELL, TAL1D, and HOX11, revealed by multiplex RT-PCR, and in 48 cases, 24 (57.1%) had chromosome abnormalities. Among the latter, numeral chromosome abnormalities and chromosome deletions accounted for 75.0% (18/24), while translocations 25.0% (6/24). The multiplex RT-PCR in combination with chromosome analysis could detect genetic abnormalities in 70% (35/50) of childhood ALL. CONCLUSIONS: Multiplex RT-PCR combined with chromosome analysis can enhance the detection rate of genetic abnormalities in childhood ALL. It provides reliable evidence for the diagnosis, classification and prognosis.

Adolescent↗