Analysis of array-based gene expression experiments is challenging particularly when multiple experiments are involved and presents challenges in data management as well. Selecting "well-measured" spots and ...
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ISBN:
(纸本)0819439444
Analysis of array-based gene expression experiments is challenging particularly when multiple experiments are involved and presents challenges in data management as well. Selecting "well-measured" spots and normalizing raw data are basic steps required for subsequent analyses, especially those involved with comparisons over a collection of experiments. Other preprocessing steps might also be needed for certain analyses. The most appropriate criteria for spot selection, for normalization, and for other data transformations depend on the experiments under study and on the questions investigated. Furthermore, comparing experiments appropriately requires knowledge of how the experiments were performed and the samples that were used in sufficient detail to understand their degree of similarity. Approaches taken in RAD to address these issues will be presented. These include the storage of raw and processed data along with history and parameter tracking and the use of ontologies to provide precise consistent experimental descriptions.
Increased sensitivity for differential mRNA expression analysis on microarrays;is rapidly becoming a serious need as the technology matures. Current techniques using direct cyanine labeled targets are effective for ex...
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ISBN:
(纸本)0819439444
Increased sensitivity for differential mRNA expression analysis on microarrays;is rapidly becoming a serious need as the technology matures. Current techniques using direct cyanine labeled targets are effective for expression analysis of abundant mRNA sources but have limited utility for analysis where mRNA quantities are limited. Tyramide signal amplification (TSA((TM))) applied to microarray detection provides dramatic improvements in sensitivity, allowing the reduction of sample sizes by as much as 200-fold. The technique includes hapten labeling of two separate RNA populations, microarray hybridization and detection of each hapten with sequential signal amplification steps. The system uses fluorescein and biotin nucleotide analogs as the hapten pair. Hybridized fluorescein and biotin labeled targets are sequentially reacted with horseradish peroxidase and cyanine 3 and cyanine 5 tyramides, resulting in the numerous depositions of these fluorophors on the array. Differential gene expression analysis of LNCaP and PC3 prostate cancer cell lines using one microgram of total RNA and TSA detection, indicates good correlation with results obtained starting with 100 micrograms (mug) of total RNA in a conventional cyanine 3 and cyanine 5 nucleotide analog labeling and detection system (i.e., the "direct method").
We describe the use of singular value decomposition in transforming genome-wide expression data from genes x arrays space to reduced diagonalized "eigengenes" x "eigenarrays" space, where the eigen...
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ISBN:
(纸本)0819439444
We describe the use of singular value decomposition in transforming genome-wide expression data from genes x arrays space to reduced diagonalized "eigengenes" x "eigenarrays" space, where the eigengenes (or eigenarrays) are unique orthonormal superpositions of the genes (or arrays). Normalizing the data by filtering out the eigengenes (and eigenarrays) that are inferred to represent additive or multiplicative noise, experimental artifacts, or even irrelevant biological processes enables meaningful comparison of the expression of different genes across different arrays in different experiments. Sorting the data according to the eigengenes and eigenarrays gives a global picture of the dynamics of gene expression, in which individual genes and arrays appear to be classified into groups of similar regulation and function, or similar cellular state and biological phenotype, respectively. After normalization and sorting, the significant eigengenes and eigenarrays can be associated with observed genome-wide effects of regulators, or with measured samples, in which these regulators are overactive or underactive, respectively.
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