title: Sensitive and precise quantification of insulin-like mRNA expression in Caenorhabditis elegans.
abstract: Insulin-like signaling regulates developmental arrest, stress resistance and lifespan in the nematode Caenorhabditis elegans. However, the genome encodes 40 insulin-like peptides, and the regulation and function of individual peptides is largely uncharacterized. We used the nCounter platform to measure mRNA expression of all 40 insulin-like peptides as well as the insulin-like receptor daf-2, its transcriptional effector daf-16, and the daf-16 target gene sod-3. We validated the platform using 53 RNA samples previously characterized by high density oligonucleotide microarray analysis. For this set of genes and the standard nCounter protocol, sensitivity and precision were comparable between the two platforms. We optimized conditions of the nCounter assay by varying the mass of total RNA used for hybridization, thereby increasing sensitivity up to 50-fold and reducing the median coefficient of variation as much as 4-fold. We used deletion mutants to demonstrate specificity of the assay, and we used optimized conditions to assay insulin-like gene expression throughout the C. elegans life cycle. We detected expression for nearly all insulin-like genes and find that they are expressed in a variety of distinct patterns suggesting complexity of regulation and specificity of function. We identified insulin-like genes that are specifically expressed during developmental arrest, larval development, adulthood and embryogenesis. These results demonstrate that the nCounter platform provides a powerful approach to analyzing insulin-like gene expression dynamics, and they suggest hypotheses about the function of individual insulin-like genes.
authors: Department of Biology and IGSP Center for Systems Biology, Duke University, Durham, North Carolina, United States of America. ryan.baugh@duke.edu
authors: L Ryan
authors: LR
authors: Baugh
authors: Nicole
authors: N
authors: Kurhanewicz
authors: Paul W
authors: PW
authors: Sternberg
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D007328:Insulin / Q000235:genetics*; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D007328:Insulin; D012333:RNA, Messenger