The Replica Set Method is a Robust, Accurate, and High-Throughput Approach for Assessing and Comparing Lifespan in C. elegans Experiments

Abstract

The advent of feeding based RNAi in Caenorhabditis elegans led to an era of gene discovery in aging research. Hundreds of gerogenes were discovered, and many are evolutionarily conserved, raising the exciting possibility that the underlying genetic basis for healthy aging in higher vertebrates could be quickly deciphered. Yet, the majority of putative gerogenes have still only been cursorily characterized, highlighting the need for high-throughput, quantitative assessments of changes in aging. A widely used surrogate measure of aging is lifespan. The traditional way to measure mortality in C. elegans tracks the deaths of individual animals over time within a relatively small population. This traditional method provides straightforward, direct measurements of median and maximum lifespan for the sampled population. However, this method is time consuming, often underpowered, and involves repeated handling of a set of animals over time, which in turn can introduce contamination or possibly damage increasingly fragile, aged animals. We have previously developed an alternative “Replica Set” methodology, which minimizes handling and increases throughput by at least an order of magnitude. The Replica Set method allows changes in lifespan to be measured for over one hundred feeding-based RNAi clones by one investigator in a single experiment- facilitating the generation of large quantitative phenotypic datasets, a prerequisite for development of biological models at a systems level. Here, we demonstrate through analysis of lifespan experiments simulated in silico that the Replica Set method is at least as precise and accurate as the traditional method in evaluating and estimating lifespan, and requires many fewer total animal observations across the course of an experiment. Furthermore, we show that the traditional approach to lifespan experiments is more vulnerable than the Replica Set method to experimental and measurement error. We find no compromise in statistical power for Replica Set experiments, even for moderate effect sizes, or when simulated experimental errors are introduced. We compare and contrast the statistical analysis of data generated by the two approaches, and highlight pitfalls common with the traditional methodology. Collectively, our analysis provides a standard of measure for each method across comparable parameters, which will be invaluable in both experimental design and evaluation of published data for lifespan studies.

TheC. elegansMyc-family of transcription factors coordinate a dynamic adaptive response to dietary restriction

“…For lifespan experiments, animals were kept from the L1 to L4 stage at 16°C, at which point FUdR was added to a final concentration of 400 μM; animals were then transferred to 20°C. Replica Set lifespan experiments were performed as detailed in (Samuelson et al 2007;Cornwell et al 2018Cornwell et al , 2022. Briefly, in Replica Set experiments, individual animals are not followed over time, but rather a large population is split across 24-well "replica" plates, and a new sub-population of animals is scored at each time point and then discarded.…”

“…Enough "replica" plates are set up at the outset of the experiment for each planned observation based on typical lifespan characteristics of the strains already established. Resulting data was fit to logistic function curve for determination of median lifespan, as previously described (Cornwell et al 2018(Cornwell et al , 2022.…”

“…Replica-set lifespan experiments for N2, eat-2(ad465), and daf-2(e1370) mutant animals kept at 20 and 25 °C post-development. Points represent observations of independent sub-populations of animals, fit with a logistic function to obtain a survival curve (Cornwell et al 2018(Cornwell et al , 2022. daf-2 mutant animals have been well-characterized as long-lived and stress-resistant.…”