Project description:The two major methods for growing C. elegans are liquid and plate culture. Differences in oxygenation and other environmental variables could affect many experimentally relevant biological processes, such as metabolism, cellular structure, and gene regulation. Worms on solid support move with deliberate sinusoidal movements, while worms in liquid thrash rapidly and almost constantly, often appearing elongated. We investigated differential gene expression under these two commonly used growth conditions. Embryos were isolated from adults grown on plates. After hatching, animals were placed either on plates or in liquid under standard conditions, and grown until young adulthood, approximately 48 hours later. RNA was isolated independently from each growth, differentially labeled with a fluorescent dye, and mixed directly for comparative hybridization to NimbleGen DNA microarrays, which contained probes designed to measure expression from all predicted C. elegans coding genes. The experiment was performed in triplicate. Analysis of the expression data yielded surprisingly few genes with differential expression, about 1%. Overall, 176 genes were downregulated significantly (p<0.01; Student's t) in liquid culture relative to plates, but of these only 102 genes were down two-fold or more. A total of 121 genes were upregulated (p<0.01. Student's t) in liquid; of these, RNA corresponding to 50 increased by at least two-fold. Surprisingly few genes directly associated with a stress response were differentially regulated in this experiment. However, the single gene most strongly upregulated in liquid is nnt-1, which encodes a mitochondrial NADP transhydrogenase, which protects against oxidative stress (Arkbladt et al., 2005 PMID: 15890626). Other molecules associated with reducing oxidation are also upregulated in liquid culture, including thioredoxin, and two glutathione-S-transferases. As a class, however, the group of genes most differentially regulated encode collagens. A total of 12 collagen genes were downregulated in liquid culture relative to plates, while none are upregulated. Other genes affected include those encoding proteins involved in ubiquitin-mediated proteolysis, Ras signaling, transcriptional regulation, ribosome function, and multiple membrane-associated proteins. The limited changes in relative RNA levels under different culture conditions provide increased confidence in comparisons made between worms grown in liquid and solid culture, and more generally between datasets obtained in different labs under slightly different culture conditions. Knowing which genes are most responsive to different culture conditions inform us about how environment affects the animal, and should be of interest for comparison to other stress and longevity global datasets.

Project description:The two major methods for growing C. elegans are liquid and plate culture. Differences in oxygenation and other environmental variables could affect many experimentally relevant biological processes, such as metabolism, cellular structure, and gene regulation. Worms on solid support move with deliberate sinusoidal movements, while worms in liquid thrash rapidly and almost constantly, often appearing elongated. We investigated differential gene expression under these two commonly used growth conditions. Embryos were isolated from adults grown on plates. After hatching, animals were placed either on plates or in liquid under standard conditions, and grown until young adulthood, approximately 48 hours later. RNA was isolated independently from each growth, differentially labeled with a fluorescent dye, and mixed directly for comparative hybridization to NimbleGen DNA microarrays, which contained probes designed to measure expression from all predicted C. elegans coding genes. The experiment was performed in triplicate. Analysis of the expression data yielded surprisingly few genes with differential expression, about 1%. Overall, 176 genes were downregulated significantly (p<0.01; Student's t) in liquid culture relative to plates, but of these only 102 genes were down two-fold or more. A total of 121 genes were upregulated (p<0.01. Student's t) in liquid; of these, RNA corresponding to 50 increased by at least two-fold. Surprisingly few genes directly associated with a stress response were differentially regulated in this experiment. However, the single gene most strongly upregulated in liquid is nnt-1, which encodes a mitochondrial NADP transhydrogenase, which protects against oxidative stress (Arkbladt et al., 2005 PMID: 15890626). Other molecules associated with reducing oxidation are also upregulated in liquid culture, including thioredoxin, and two glutathione-S-transferases. As a class, however, the group of genes most differentially regulated encode collagens. A total of 12 collagen genes were downregulated in liquid culture relative to plates, while none are upregulated. Other genes affected include those encoding proteins involved in ubiquitin-mediated proteolysis, Ras signaling, transcriptional regulation, ribosome function, and multiple membrane-associated proteins. The limited changes in relative RNA levels under different culture conditions provide increased confidence in comparisons made between worms grown in liquid and solid culture, and more generally between datasets obtained in different labs under slightly different culture conditions. Knowing which genes are most responsive to different culture conditions inform us about how environment affects the animal, and should be of interest for comparison to other stress and longevity global datasets. Total RNA was extracted from young adult hermaphrodite C. elegans grown either in s-basal liquid cultures or nematode growth medium plates under standard conditions.

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Caenorhabditis elegans

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