Technical Analysis of Fundulus heteroclitus cDNA Microarrays, experiment B
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ABSTRACT:
ORGANISM(S): Fundulus heteroclitus
PROVIDER: GSE12898 | GEO | 2008/11/21
SECONDARY ACCESSION(S): PRJNA114265
REPOSITORIES: GEO
Similar Datasets
Project description:In the teleost fish Fundulus heteroclitus there is extensive variation in gene expression among individuals within and between populations. Accurate measures of the variation in mRNA expression using microarrays can be confounded by technical variation, which includes variation in RNA isolation procedures, day of hybridization and methods used to amplify and dye label RNA for hybridization. In this manuscript we analyze the relationship between the amount of mRNA and the fluorescent signal from the microarray hybridizations demonstrating that for a wide-range of mRNA concentrations the fluorescent signal is a linear function of the amount of mRNA. Additionally, the separate isolation, labeling or hybridization of RNA does not add significant amounts of variation in microarray measures of gene expression. However, single or double rounds of amplification for labeling do have small but significant affects on 10% of genes, but this source of technical variation is easy to avoid. To examine both technical and stochastic biological variation, mRNA expression was measured from the same five individuals over a six-week time course. There were few, if any, meaningful differences in gene expression among time points. Thus, microarray measures using standard laboratory procedures can be precise and quantitative and are not subject to significant random biological noise.
2008-11-21 | GSE12858 | GEO
Project description:Analysis of the relationship between the amount of mRNA and the fluorescent signal from the microarray hybridizations. In addition, we measured expression for differences in RNA labeling and for the day on which RNA is prepared. Keywords: Gene expression Two-condition experiment, number of rounds of RNA amplification and the day on which it was prepared. Four replicates per gene per array.
2010-05-15 | E-GEOD-12898 | biostudies-arrayexpress
Project description:In the teleost fish Fundulus heteroclitus there is extensive variation in gene expression among individuals within and between populations. Accurate measures of the variation in mRNA expression using microarrays can be confounded by technical variation, which includes variation in RNA isolation procedures, day of hybridization and methods used to amplify and dye label RNA for hybridization. In this manuscript we analyze the relationship between the amount of mRNA and the fluorescent signal from the microarray hybridizations demonstrating that for a wide-range of mRNA concentrations the fluorescent signal is a linear function of the amount of mRNA. Additionally, the separate isolation, labeling or hybridization of RNA does not add significant amounts of variation in microarray measures of gene expression. However, single or double rounds of amplification for labeling do have small but significant affects on 10% of genes, but this source of technical variation is easy to avoid. To examine both technical and stochastic biological variation, mRNA expression was measured from the same five individuals over a six-week time course. There were few, if any, meaningful differences in gene expression among time points. Thus, microarray measures using standard laboratory procedures can be precise and quantitative and are not subject to significant random biological noise. mRNA expression was measured using microarrays where each array had four spatially separated replicates per gene. The 384 F. heteroclitus cDNA microarrays were printed using 55 control genes and 329 cDNAs which encode essential proteins for cellular metabolism. To test for the relationship between fluorescence and the quantity of RNA, five concentrations of fluorescently labeled RNA were used: 1.2 to 700 pmol of Cy3 or Cy5. A 15 µl hybridization using the 384 cDNA array corresponds to 0.09 to 47 µM of Cy dye. Cy5 dye labeled RNA was used at concentrations 18% less than Cy3 because the Cy5 dye is a more efficient fluorophore (greater fluorescence per photon) than the Cy3 dye. The average of eight fluorescence values for each gene was normalized to the original concentration of RNA added.
2010-05-15 | E-GEOD-12858 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below.
2008-11-21 | GSE13687 | GEO
Project description:This SuperSeries is composed of the following subset Series: GSE12858: Technical Analysis of Fundulus heteroclitus cDNA Microarrays, experiment A GSE12898: Technical Analysis of Fundulus heteroclitus cDNA Microarrays, experiment B Refer to individual Series
2010-05-15 | E-GEOD-13687 | biostudies-arrayexpress
Project description:Most studies that examine the ontogeny of lymphoid organ development in teleostean fishes use species of interest to aquaculture or genetic research and, to date, have focused strictly on marine or freshwater species. The mummichog, Fundulus heteroclitus, also known as the estuarine killifish, is a unique model for studies on developmental immunobiology, because it is euryhaline, has a high degree of thermal tolerance, and has a unique reproductive strategy. Embryonic and larval mummichogs were examined for the ontogeny of lymphoid tissue development. The first lymphoid organ to appear was the head kidney at 1 dph, followed by the spleen at 1 wph, and then the thymus at 3 wph. Rag-1 was partially cloned and sequenced and shown to be highly conserved among other vertebrate Rag-1 genes. Using QT-PCR to monitor the temporal expression of Rag-1, it was shown to reach a maximum intensity at 3 and 4 wph and then to drop to pre-2-wph levels. Overall, this study suggests that juvenile mummichogs do not possess the ability to mount T- or B-cell responses until some time after 5 wph. Even though the estuarine killifish tolerates a wide range of salinities, the developmental patterns of lymphoid tissues are similar to what has been reported for strictly marine (stenohaline) teleosts. Thus, the mummichog should be a convenient model for understanding the developmental immunobiology of most marine teleosts.
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