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.
____________________________
Publisher: Public Library of Science
Date Published: 22-March-2011
Author(s): Baugh L., Kurhanewicz N., Sternberg P.
Link: https://doi.org/10.1371/journal.pone.0018086