Assessing age-dependent effects of vhp-1 knock-down on gene expression
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ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
PROVIDER: GSE82238 | GEO | 2016/06/04
SECONDARY ACCESSION(S): PRJNA324413
REPOSITORIES: GEO
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Project description:Purpose: The objective of this study was to identify genes whose expression changes significantly between the L4 larval stage (Day 0 of adulthood) and the sixth day of adulthood (Day 6) in C. elegans. Methods: Wild type N2 animals were synchronized by sodium hypochlorite treatment and dropped as L1 larvae to NGM RNAi plates containing 2mM IPTG, 100 mg/mL carbenicillin and seeded with E. coli strain HT115 harboring the L4440 plasmid (vector only control for feeding-based RNAi experiments). Animals were maintained on these plates at 20˚C until the L4 larval stage when half of the cohort was harvested for total RNA (brief description to follow). The other half of the cohort was transferred to new NGM RNAi plates containing 2mM IPTG, 100 mg/mL carbenicillin, 25ug/mL FUDR and seeded with E. coli strain HT115 harboring the L4440 plasmid. C. elegans were maintained on these plates at 20˚C until they reached the sixth day of adulthood when they were harvested for total RNA. To isolate RNA, worms were washed off of plates in M9 buffer, pelletted by centrifugation and resuspended in Trizol. Following a phenol/chloroform extraction, total RNA was precipitated in isopropanol, washed in 70% ethanol, and resuspended in RNase-free H2O. Total RNA was treated with DNase and reisolated using Qiagen RNeasy columns. RNA was isolated from two independent cohorts of L4 animals and three independent cohorts of Day 6 adults. rRNA was depleted from total RNA using Ribo-Zero and libraries were prepared by strand-specific cDNA synthesis for Illumina HiSeq sequencing. Following quality control steps to check for ribosomal depletion, contamination, and leftover adapter sequences, sequences were trimmed and alinged to the C. elegans genome using TopHat2. Alignments were then run with featureCounts then normalized using edgeR's calcNormFactors then transformmed using limma's voom. Changes in gene expression were identified by performing a differential expression analysis using DESeq2 on the Igenbio ERGO platform (Igenbio, Inc., Chicago, Il). Results: Results from DESeq2 were filtered for statistically significant changes in expression by accepting only those changes with a q-value of less than 0.05. As compared to expression at the L4 larval stage, at Day 6 of adulthood we found there to be 1441 genes that were upregulated by more than 5-fold and 2660 genes were downregulated by more than 5-fold. Conclusions: Aging is commensurate with significant and substantial changes in gene expression in C. elegans. By the time adults enter the post-reproductive phase of their life, we found that approximately 4000 genes are differentially expressed as compared to their expression levels in L4 larvae. While some of these changes in expression may drive the aging process, others may protect animals from age-associated damage.
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