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 by bleaching from adults grown for several generations on plates. After hatching in M9 media, animals were placed either on plates seeded with OP50 or in liquid supplemented with HB101 under standard conditions. Both cultures were grown until worms reached young adulthood, approximately 48 hours later. Total RNA was isolated independently from each growth, differentially labeled with a fluorescent dye, and mixed directly for comparative hybridization to 4-plex NimbleGen DNA microarrays, which contained 72,000 probes, which covered all predicted C. elegans protein-coding genes (minimum 3 probes per gene). 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). 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 tells us how environment affects the animal, and should be of interest for comparison to other stress and longevity global datasets. The data is being prepared for formal publication, and is available via GEO accession number GSE21526.
Arkblad EL, Tuck S, Pestov NB, Dmitriev RI, Kostina MB, Stenvall J, Tranberg M, Rydström J. (2005). A Caenorhabditis elegans mutant lacking functional nicotinamide nucleotide transhydrogenase displays increased sensitivity to oxidative stress. Free Radic. Biol. Med. 38, 1518-25.
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