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The Replica Set Method is a Robust, Accurate, and High-Throughput Approach for Assessing and Comparing Lifespan in C. elegans Experiments
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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9261357/ https://www.ncbi.nlm.nih.gov/pubmed/35821830 http://dx.doi.org/10.3389/fragi.2022.861701 |
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author | Cornwell, Adam Llop, Jesse R. Salzman, Peter Rasmussen, Niels Thakar, Juilee Samuelson, Andrew V. |
author_facet | Cornwell, Adam Llop, Jesse R. Salzman, Peter Rasmussen, Niels Thakar, Juilee Samuelson, Andrew V. |
author_sort | Cornwell, Adam |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-9261357 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-92613572022-07-11 The Replica Set Method is a Robust, Accurate, and High-Throughput Approach for Assessing and Comparing Lifespan in C. elegans Experiments Cornwell, Adam Llop, Jesse R. Salzman, Peter Rasmussen, Niels Thakar, Juilee Samuelson, Andrew V. Front Aging Aging 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. Frontiers Media S.A. 2022-04-28 /pmc/articles/PMC9261357/ /pubmed/35821830 http://dx.doi.org/10.3389/fragi.2022.861701 Text en Copyright © 2022 Cornwell, Llop, Salzman, Rasmussen, Thakar and Samuelson. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Aging Cornwell, Adam Llop, Jesse R. Salzman, Peter Rasmussen, Niels Thakar, Juilee Samuelson, Andrew V. The Replica Set Method is a Robust, Accurate, and High-Throughput Approach for Assessing and Comparing Lifespan in C. elegans Experiments |
title | The Replica Set Method is a Robust, Accurate, and High-Throughput Approach for Assessing and Comparing Lifespan in C. elegans Experiments |
title_full | The Replica Set Method is a Robust, Accurate, and High-Throughput Approach for Assessing and Comparing Lifespan in C. elegans Experiments |
title_fullStr | The Replica Set Method is a Robust, Accurate, and High-Throughput Approach for Assessing and Comparing Lifespan in C. elegans Experiments |
title_full_unstemmed | The Replica Set Method is a Robust, Accurate, and High-Throughput Approach for Assessing and Comparing Lifespan in C. elegans Experiments |
title_short | The Replica Set Method is a Robust, Accurate, and High-Throughput Approach for Assessing and Comparing Lifespan in C. elegans Experiments |
title_sort | replica set method is a robust, accurate, and high-throughput approach for assessing and comparing lifespan in c. elegans experiments |
topic | Aging |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9261357/ https://www.ncbi.nlm.nih.gov/pubmed/35821830 http://dx.doi.org/10.3389/fragi.2022.861701 |
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