The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Summary

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

Abstract

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

Introduction

One of the most transformative technological advancements towards understanding the genetic basis of aging was the development of feeding-based RNAi in C. elegans¹; prior to the experimental use of RNAi, many phenotypes of aging were not genetically tractable. Feeding-based RNAi is achieved through the production of dsRNA within E. coli that matches an endogenous C. elegans mRNA: IPTG induces bidirectional transcription across an insert of either C. elegans cDNA or a portion of an open reading frame within a plasmid². When C. elegans feed upon intact E. coli,....

Protocol

1. Traditional Method for Scoring C. elegans Longevity

1. Preparation of reagents
   1. Identify genes to be inactivated via feeding-based RNAi. Purchase transformed stocks of HT115 E. coli² containing the RNAi clone of interest. Alternatively, subclone the cDNA of the gene of interest into the multicloning site of the L4440 plasmid.
      NOTE: HT115 is an RNase III-deficient E. coli strain with IPTG-inducible T7 polymerase activity, which is used to prevent degradation of dsRNA within the bacteria. For lifespan studies that do not use feeding-based RNAi, either HT115 or OP50 E. ....

Results

In the development of any new methodology, it is imperative that the new method recapitulates accepted results from previous approaches and meets the standard within a field. We have previously shown empirically that the Replica Set and traditional methods for assaying C. elegans lifespan produce similar results²⁰. Wild-type C. elegans (N2) maintained at 20 °C typically live between 20 and 25 days, which we observed with both the traditional (

Discussion

Both the traditional and replica set methods require the synchronization of chronologically aged animals. We include a method that synchronizes animals using hypochlorite treatment of gravid adults, where only fertilized eggs with the gravid adult survive treatment. These embryos hatch in liquid suspension and developmentally arrest at the first larval stage (L1). After seeding L1 animals onto food (e.g. E. coli expressing dsRNA to a gene of interest), animals resume development. Synchronizing L1 animal.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IPTG (isopropyl beta-D-1-thigalactopyranoside)	Gold Bio	12481C100
FuDR (5-Fluoro-2'-deoxyuridine)	Alfa Aesar	L16497
24 Well Culture Plates	Greiner Bio-One	#662102
Retangular non-treated single-well plate, 128x86mm	Thermo-Fisher	242811
600 µL 96-well plates	Greiner Bio-One	#786261
2mL 96-well plates	Greiner Bio-One	#780286
Air-permeable plate seal	VWR	60941-086
96-pin plate replicator	Nunc	250520
bacto-peptone	VWR	90000-368
bacteriological agar	Affymetrix/USB	10906
C. elegans RNAi clone library in HT115 bacteria- Ahringer	Source Bioscience	C. elegans RNAi Collection (Ahringer)	See also Kamath et. al, Nature 2003.
C. elegans RNAi clone library in HT115 bacteria- Vidal	Source Bioscience	C. elegans ORF-RNAi Resource (Vidal)	See also Rual et. al, Genome Research 2004. This library is also available from Dharmacon.
WormLife- Software for Replica Set Survival Analysis	Samuelson Lab	N/A	https://github.com/samuelsonlab-urmc/wormlife
L4440 Empty Vector Plasmid	Addgene	1654	https://www.addgene.org/1654/
Wormbase			http://www.wormbase.org/
OASIS			https://sbi.postech.ac.kr/oasis2/
Graphpad Prism			https://www.graphpad.com/scientific-software/prism/