Biomedical subjects

Xujing Wang

Publications and source records attributed to Xujing Wang.

5 recordsLinked to original sources

PubMed2003

Quantitative quality control in microarray experiments and the application in data filtering, normalization and false positive rate prediction.

Data preprocessing including proper normalization and adequate quality control before complex data mining is crucial for studies using the cDNA microarray technology. We have developed a simple procedure that integrates data filtering and normalization with quantitative quality control of microarray experiments. Previously we have shown that data variability in a microarray experiment can be very well captured by a quality score q(com) that is defined for every spot, and the ratio distribution depends on q(com). Utilizing this knowledge, our data-filtering scheme allows the investigator to decide on the filtering stringency according to desired data variability, and our normalization procedure corrects the q(com)-dependent dye biases in terms of both the location and the spread of the ratio distribution. In addition, we propose a statistical model for false positive rate determination based on the design and the quality of a microarray experiment. The model predicts that a lower limit of 0.5 for the replicate concordance rate is needed in order to be certain of true positives. Our work demonstrates the importance and advantages of having a quantitative quality control scheme for microarrays.

Algorithms↗

PubMed2003

Use of a three-color cDNA microarray platform to measure and control support-bound probe for improved data quality and reproducibility.

Construction methodologies for cDNA microarrays lack the ability to determine array integrity prior to hybridization, leaving the array itself a source of uncontrolled experimental variation. We solved this problem through development of a three-color cDNA array platform whereby printed probes are tagged with fluorescein and are compatible with Cy3 and Cy5 target labeling dyes when using confocal laser scanners possessing narrow bandwidths. Here we use this approach to: (i) develop a tracking system to monitor the printing of probe plates at predicted coordinates; (ii) define the quantity of immobilized probe necessary for quality hybridized array data to establish pre-hybridization array selection criteria; (iii) investigate factors that influence probe availability for hybridization; and (iv) explore the feasibility of hybridized data filtering using element fluorescein intensity. A direct and significant relationship (R2 = 0.73, P < 0.001) between pre-hybridization average fluorescein intensity and subsequent hybridized replicate consistency was observed, illustrating that data quality can be improved by selecting arrays that meet defined pre-hybridization criteria. Furthermore, we demonstrate that our three-color approach provides a means to filter spots possessing insufficient bound probe from hybridized data sets to further improve data quality. Collectively, this strategy will improve microarray data and increase its utility as a sensitive screening tool.

Color↗

PubMed2003

Three color cDNA microarrays: quantitative assessment through the use of fluorescein-labeled probes.

Gene expression studies using microarrays have great potential to generate new insights into human disease pathogenesis, but data quality remains a major obstacle. In particular, there does not exist a method to determine prior to hybridization whether an array will yield high quality data, given good study design and target preparation. We have solved this problem through development of a three-color cDNA microarray platform where printed probes are fluorescein labeled, but are spectrally compatible with Cy3 and Cy5 dye-labeled targets when using confocal laser scanners possessing narrow bandwidths. This approach enables prehybridization evaluation of array/spot morphology, DNA deposition and retention and background levels. By using these measurements and the intra-slide coefficient of variation for fluorescence intensity we show that slides in the same batch are not equivalent and measurable prehybridization parameters can be predictive of hybridization performance as determined by replicate consistency. When hybridizing target derived from two cell lines to high and low quality replicate pairs (n = 50 pairs), a direct and significant relationship between prehybridization signal-to-background noise and post-hybridization reproducibility (R2 = 0.80, P < 0.001) was observed. We therefore conclude that slide selection based upon prehybridization quality scores will greatly benefit the ability to generate reliable gene expression data.

Carbocyanines↗

PubMed2002

Membrane dielectric changes indicate induced apoptosis in HL-60 cells more sensitively than surface phosphatidylserine expression or DNA fragmentation.

The specific membrane capacitance and conductivity of mammalian cells, which reflect their surface morphological complexities and membrane barrier functions, respectively, have been shown to respond to cell physiologic and pathologic changes. Here, the effects of induced apoptosis on these membrane properties of cultured human promyelocytic HL-60 cells are reported. Changes in membrane capacitance and conductivity were deduced from measurements of cellular dielectrophoretic crossover frequencies following treatment with genistein (GEN). The apparent specific cell membrane capacitance of HL-60 cells fell from an initial value of 17.6+/-0.9 to 9.1+/-0.5 mF/m(2) 4 h after treatment. Changes began within minutes of treatment and preceded both the externalization of phosphatidylserine (PS), as gauged by the Annexin V assay, and the appearance of a sub-G1 cell subpopulation, as determined through ethidium bromide staining of DNA. Treatment by the broad spectrum caspase inhibitor N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethyketone (zVAD-fmk) did not prevent these early cell membrane dielectric responses, suggesting that the caspase system was not involved. Although membrane conductivity did not alter during the first 4 h of GEN treatment, it rose significantly and progressively thereafter. Finally, as the barrier function failed and the cells became necrotic, it increased by many orders of magnitude. The effective membrane capacitance and conductivity findings serve to focus attention on the membrane as a site for early participation in apoptosis. In conjunction with our prior reports of the use of dielectric methods for cell manipulation and separation, these results demonstrate that dielectrophoretic technologies should be applicable to the rapid detection, separation, and quantification of normal, apoptotic, and necrotic cells from cell mixtures.

Apoptosis↗

PubMed2002

Conditions to ensure competitive hybridization in two-color microarray: a theoretical and experimental analysis.

We derived a theoretical model that explains certain biases observed in the two-color microarray hybridization experiments reported in the literature. We show that true competition is achieved only when the hybridization kinetics of the two differentially labeled probes are the same. If the hybridization kinetics of the two differentially labeled probes is different, which can occur when the labeling and hybridization conditions for the two probes are dissimilar, then differential expression observed becomes a function of the amount of the target (i.e., DNA spotted on the slide). We use this model to validate the microarray methodology by determining the differential expression of four select Arabidopsis genes and two human genes (beta-actin and GAPDH) as a function of the amount of target arrayed. We show through both modeling and experiments that the rate constants for Cy5- and Cy3-labeled probes are the same under our exrimental conditions. Therefore, the target concentrations need not greatly exceed the probe concentration. It is obvious from the data presented that a simple treatment of an individual hybridization rate calculation does notfully describe what is occuring in today's complex, multispecies experiments. The method of validation is easily implemented to ensure data reliability by two-color microarray.

Arabidopsis↗