Funding for this work described in this manuscript was provided by: the University of Rochester Office of The Provost and the School of Medicine and Dentistry Dean&#39;s Office via the Health Sciences Center for Computational Innovation (HSCCI); the Ellison Medical Foundation New Scholars in Aging Fellowship (AG-NS-0681-10) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

<ol>
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The authors declare that they have no competing financial interests.

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 animals by hypochlorite treatment of gravid adults has the advantage that the leftover unseeded L1 animals can be frozen and stored indefinitely in liquid nitrogen or a -80 &#176;C freezer. In this way, a sample of each strain at the time of the experimental setup is preserved, creating a valuable resource for future studies and improving reproducibility. However, while hypochlorite treatment of gravid adult animals is a common way to obtain synchronized animals for aging research22, the L1 arrest is a starvation response. Thus, some laboratories prefer either to allow hatching to occur on plates, or to forgo hypochlorite treatment altogether and allow a few gravid adults to lay eggs for several hours (i.e. an egg lay). In the latter case, parents are removed and the progeny lifespan is followed. To the best of our knowledge, no obvious differences in lifespan have been reported between animals that were synchronized in M9, hatched on plates, or progeny from an egg lay. However, given that changes in nutrient availability are closely linked to changes in lifespan, there is precedence that specific genetic backgrounds could produce different outcomes between these synchronization approaches. A more careful analysis is required to resolve this theoretical concern.
Regardless of the method used to synchronize the starting population, steps must be taken to either prevent progeny production or to separate the synchronized starting population from future progeny. In our protocol, we outline how to use FUdR to prevent progeny production, as separating animals is not a viable option for the replica set method. It is also possible to prevent progeny production genetically through the use of a feminized genetic background (e.g. fer-15(b26);fem-1(hc17), which is a temperature-dependent sterile strain23). However, neither is without shortcomings: the use of genetic backgrounds can complicate subsequent analysis, and in some genetic backgrounds FUdR can alter longevity24,25,26.
As an alternative to preventing progeny production through chemical or genetic means, adult animals can be periodically moved to fresh RNAi plates to isolate them from their progeny. This simplifies background considerations at the expense of throughput. Periodically moving animals to fresh food has the additional advantages of preventing possible starvation and renewing exposure to dsRNA. However, some genetic interactions that influence lifespan were only discovered when progeny production was inhibited: early analysis of the TGF&#946; pathway for a lifespan phenotype erroneously concluded that decreased TGF&#946; signaling influenced C. elegans dauer formation but not aging27,28. However, a follow up study that used FUdR revealed that decreased TGF&#946; signaling increased longevity through insulin signaling29. Why did earlier studies fail to see increased lifespan in TGF&#946; mutant animals? TGF&#946; pathway mutations produce a slight egg laying defect (egl) and extend reproductive longevity, which causes internal hatching of progeny later in life that kills the parent. It is likely that the long-lived TGF&#946; mutant animals appeared to have a normal lifespan because of the egl phenotype killed the animals around the time when wild-type animals normally die. This might be relevant to other genetic pathways linked to DR, as starved wild-type animals also manifest an egl phenotype, perhaps as an adaptive survival advantage to progeny under conditions of low food. This highlights the underlying complexity in adaptive responses animals undergo under stress conditions, and the need for careful analysis and consideration when designing lifespan experiments.
In designing and conducting lifespan experiments by either method it is critical to avoid bias. Experiments must be conducted in a double-blind manner: how samples were previously scored at previous time points and the identity of a test condition must be unknown to the experimenter. Furthermore, it is always necessary to include both positive and negative controls; in the case of the replica set method, these are randomly inserted into a 24-well plate. E. coli expressing a plasmid that does not contain an insert with the sequence corresponding to the C. elegans genome is the empty vector negative control (i.e. &#34;L4440&#34;- See Materials). Positive controls are dependent on the specific nature of an experiment. For instance, daf-2 encodes the C. elegans insulin/IGF-1 receptor, and daf-2 inactivation via feeding-based RNAi robustly increases lifespan at least two-fold in wild-type animals27. Thus daf-2(RNAi) might serve as a positive control when looking for gene inactivations that increase lifespan. Conversely, daf-16 encodes the FOXO transcription factor orthologue30. DAF-16 is an essential component of many longevity paradigms and wild-type animals (N2) treated with daf-16(RNAi) are short lived and show signs of progeria31.
The primary advantages of the traditional longitudinal lifespan approach are that it is very well established, and experiments are easy to set up. Relatively few animals are needed on just a few plates for each test condition. Thus, strains that grow poorly or require balancers to propagate can be easily tested. The traditional approach is highly adaptable and can be used with any one of the available approaches for handling progeny production, including treatment with FUdR, crossing into a feminized genetic background, or periodically moving adult animals to new plates during the egg-laying period. While moving animals greatly reduces throughput, working with a mutant background is never ideal, and although FUdR does not alter wild-type lifespan32,33,34, it can affect lifespan and age related phenotypes in some genetic backgrounds35,25,26. Note that the presence of males, even with the use of FUdR, will significantly shorten hermaphrodite lifespan13, thus a plate that contained males after the hermaphrodites reached L4 is unusable. Likewise, analysis through the Kaplan-Meier estimator and associated curves, and the log-rank test, is well established for mortality data. However, there are several disadvantages to the traditional lifespan analysis. Repeated handling of plates (i.e. exposing the plates to air) facilitates the introduction of airborne fungal contamination. Additionally, repeated poking can damage or kill animals, especially as the population advances in age and become fragile. Older animals become largely paralyzed and mired in E. coli, while E. coli becomes an opportunistic pathogen (colonizing the lumen and packing the pharynx)36. Very old living animals can only be identified by subtle head movements. Thus, it is easy to classify a decrepit live animal as dead. Lastly, the traditional approach is limited by throughput.
The Replica Set method is high throughput and quantitative. However, the disadvantage of this method is a larger investment of time and resources in the initial set up. A moderately large experiment to examine 100 RNAi clones over 20 time points requires 30,000 L1 animals (where approximately 15 animals are examined per RNAi clone per time point), which while easy for most strains, can be problematic in some cases. For instance, without a large-particle sorter (&#34;worm sorter&#34;) strains that must be maintained with balancers or transgenic lines carrying a poorly transmitted extra-chromosomal array cannot be easily examined by this method. A second disadvantage is that progeny production must be inhibited, which requires the use of FUdR or a feminized genetic background. Finally, one must know the length of time the assay will run, as one must prepare a replica set for each time point at the beginning of the experiment. However, the advantages of this method are numerous. Foremost, scoring viability is much faster and one can easily follow animals over 100 test conditions simultaneously (i.e. RNAi clones). Since a replica set is only scored once then discarded, there is no repeated handling of plates or poking of animals, which minimizes the likelihood of fungal contamination and eliminates mortality caused by the occasional rough prodding with a worm pick. Furthermore, the addition of liquid to the well greatly facilitates scoring. Freeing old animals from the plate and surrounding bacteria assists in allowing subtle head movements to be more easily scored. Addition of liquid also provides the opportunity to measure thrashing rates as a measure of fitness (e.g. healthspan8,37).
Aging is a complex phenomenon involving multiple causal mechanisms which require use of systems biological approaches to unravel. These approaches often incorporate data-driven modeling, using large volumes of genomic/transcriptomic data, and require complementary robust and high-throughput methods to measure lifespan and healthspan. The high-throughput Replica Set method will allow comparison of many RNAi clones longitudinally, while minimizing batch effects and technical errors, thus facilitating the development of dynamic models that can infer the interactions between causal pathways in a quantitative manner. Additionally, integration of several genome-wide genomics approaches with the Replica Set method is feasible because a large population of age-synchronized animals is divided into a number of small populations.
Other methods have been previously developed to improve the throughput of C. elegans lifespan experiments, often focusing on adapting the traditional longitudinal approach (i.e. following the same set of animals over time) to automated observation and recording using common flatbed scanners38,39, or more specialized equipment such as microfluidic plates40. The scanner-based approaches use light as a stimulus and compare sequentially captured images to determine alive/dead status based on movement for multiple plates at one time; while such approaches do not require proprietary scientific hardware, time involved in setting up the workflows may be substantial depending on the desired scale. Alternatively, lifespan experiments in custom microfluidic devices allow for in-depth phenotypic characterization of single animals over time, and without treatment to prevent progeny, but necessitate fabrication of the microfluidic plates and acquisition of associated microfluidic pumps and imaging equipment. In contrast, the Replica Set method, in combination with the software detailed here, allows greatly improved throughput using tools that are already common in laboratories working with C. elegans.
The WormLife software will be improved in the future to offer easier access to the statistical comparison, and compatibility with additional platforms. The most up-to-date documentation for the software can be found at the GitHub page, including installation instructions for platforms on which the software has been tested. A web-based version will also be developed to enable convenient access without the need to install any software.
In summary, the combination of the Replica Set method and the freely available software detailed here provides a powerful platform for improving the throughput and robustness of lifespan experiments and a broad range of survival-based assays (e.g. stress tolerance, toxicology studies, healthspan, etc.). Particularly when combined with functional genomics, this approach leverages the many benefits of the metazoan model system C. elegans for deciphering the myriad of genetic interactions that contribute to the progression of aging.

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, dsRNA produced by bacteria is transported from the lumen into intestinal cells via the SID-2 transmembrane protein3, and then distributed through the rest of the animal via SID-14. Within each cell, exogenous dsRNA is processed by the Dicer complex into siRNA, which interact with a mature mRNA via complementary base pairing to create a new siRNA-mRNA duplex. This duplex is recognized by the RISC complex and cleaved, thereby degrading the endogenous mRNA5. Thus, by merely changing the plasmid insert, one can inactivate the function of nearly any gene within the C. elegans genome. This discovery led to the creation of several large feeding-based RNAi libraries- collections of transformed E. coli stocks that can be combined to achieve coverage of approximately 86% of known C. elegans genes6,7.
Since the advancement of feeding-based RNAi, comprehensive screens in C. elegans have led to the discovery of more than 900 genes that alter lifespan when inactivated (as evidenced by the RNAi-phenotype associations curated in WormBase), which we refer to as gerogenes. A role for the majority of gerogenes in longevity control was discovered through feeding-based RNAi in just a few seminal reports (see Figure 1A and Supplemental File 1 for details). In some cases, these gerogenes have been identified based on measuring the viability at a single or a few time points, which fails to provide a quantifiable measure of the change in lifespan with RNAi treatment. In other cases, these genes have been quantitatively assessed for changes in lifespan, as well as additional age-associated phenotypes. For instance, we previously identified 159 genes that were necessary for both normal and increased lifespan of animals with decreased insulin/IGF-1 signaling, and quantified changes in healthspan. Of these, 103 gene inactivations result in a progeric phenotype, as loss resulted in one or more signs of premature aging8.
While some gerogenes have been associated with 100 or more studies (e.g. daf-16, daf-2, sir-2.1), over 400 gerogenes have 10 or fewer citations (Figure 1B, and Supplemental File 2). Thus, while comprehensive feeding-based RNAi screens have discovered and cursorily characterized hundreds of putative gerogenes, how these genes function in longevity control, and the genetic interrelationships between these gene products remain poorly studied. Full longitudinal analysis for age-associated phenotypes is a prerequisite for identifying genetic interactions between gerogenes (e.g. epistatic interactions, asynthetic interactions, etc.). Gaining deeper insight into the genetic interrelationships between gerogenes requires a high-throughput quantitative method, which also leverages the advantages of feeding-based RNAi.
The most common surrogate measure of aging is lifespan. The traditional approach for measuring C. elegans mortality tracks the deaths of individual animals over time within a small population sample. A relatively small number of animals are followed over time and periodically are gently prodded with either a platinum wire or eyelash, with movement as an indicator of viability (Figure 2A). This method has been widely used, as it provides straightforward, direct measurements of the average and the maximum lifespan. However, this traditional method is time consuming and relatively low-throughput, which limits the number of animals and conditions that can simultaneously be measured in a controlled manner. A recent simulation study found that many C. elegans lifespan studies do not assay a large enough number of animals to be able to reliably detect small changes between conditions9. Furthermore, this traditional method involves repeatedly handling the same cohort of animals over time, which in turn can introduce contamination, and can damage or kill increasingly fragile, aged animals.
We have developed an alternative &#34;Replica Set&#34; methodology for measuring C. elegans lifespan. To this end, a large population of age-synchronized, isogenic animals are divided into a number of small populations (or replicas). Enough replica samples are generated to cover each time point in the planned experiment. At each observation time point, one of the replicas is scored for the number of living, dead and censored animals, then animals within that replicate are discarded. Thus, over the expected lifespan of the population as a whole, a series of independent subpopulations are periodically sampled (Figure 2B). In using replica sets there is no repeated prodding of animals and no repeated exposure to potential environmental contamination. The viability observed at the one-time point is completely independent of every other observation, which minimizes handling and increases throughput by at least an order of magnitude. This has allowed us to quantitate changes in lifespan for hundreds of RNAi clones simultaneously8,10.
Here we present detailed protocols for conducting C. elegans lifespan via both the Replica Set and traditional methods for scoring C. elegans longevity. We demonstrate that similar results are obtained between the methods. We have developed software to assist in the graphical analysis of lifespan data generated through either approach, which we freely provide under a GPL V3 license (See Table of Materials). &#34;WormLife&#34; is written in R11, and includes a graphical user interface (GUI) for plotting data, which has been tested in Mac OS and Linux. Lastly, we compare and contrast the limitations of each method and highlight other considerations when choosing between approaches to measure quantitative changes in C. elegans lifespan.

<table><tbody><tr><td>IPTG (isopropyl beta-D-1-thigalactopyranoside)</td><td>Gold Bio</td><td>12481C100</td><td></td></tr><tr><td>FuDR (5-Fluoro-2&#039;-deoxyuridine)</td><td>Alfa Aesar</td><td>L16497</td><td></td></tr><tr><td>24 Well Culture Plates</td><td>Greiner Bio-One</td><td>#662102</td><td></td></tr><tr><td>Retangular non-treated single-well plate, 128x86mm</td><td>Thermo-Fisher</td><td>242811</td><td></td></tr><tr><td>600 &amp;micro;L 96-well plates</td><td>Greiner Bio-One</td><td>#786261</td><td></td></tr><tr><td>2mL 96-well plates</td><td>Greiner Bio-One</td><td>#780286</td><td></td></tr><tr><td>Air-permeable plate seal</td><td>VWR</td><td>60941-086</td><td></td></tr><tr><td>96-pin plate replicator</td><td>Nunc</td><td>250520</td><td></td></tr><tr><td>bacto-peptone</td><td>VWR</td><td>90000-368</td><td></td></tr><tr><td>bacteriological agar</td><td>Affymetrix/USB</td><td>10906</td><td></td></tr><tr><td>C. elegans RNAi clone library in HT115 bacteria- Ahringer</td><td>Source Bioscience</td><td>C. elegans RNAi Collection (Ahringer)</td><td>See also Kamath et. al, Nature 2003.</td></tr><tr><td>C. elegans RNAi clone library in HT115 bacteria- Vidal</td><td>Source Bioscience</td><td>C. elegans ORF-RNAi Resource (Vidal)</td><td>See also Rual et. al, Genome Research 2004. This library is also available from Dharmacon.</td></tr><tr><td>WormLife- Software for Replica Set Survival Analysis</td><td>Samuelson Lab</td><td>N/A</td><td>https://github.com/samuelsonlab-urmc/wormlife</td></tr><tr><td>L4440 Empty Vector Plasmid</td><td>Addgene</td><td>1654</td><td>https://www.addgene.org/1654/</td></tr><tr><td>Wormbase</td><td></td><td></td><td>http://www.wormbase.org/&amp;nbsp;</td></tr><tr><td>OASIS</td><td></td><td></td><td>https://sbi.postech.ac.kr/oasis2/&amp;nbsp;</td></tr><tr><td>Graphpad Prism</td><td></td><td></td><td>https://www.graphpad.com/scientific-software/prism/&amp;nbsp;</td></tr></tbody></table>

the replica set method: a high-throughput approach to quantitatively measure caenorhabditis elegans lifespan

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.

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 &#176;C typically live between 20 and 25 days, which we observed with both the traditional (Figure 4A, black line) and Replica Set approach (Figure 4B, black). Thus, both methods reasonably approximate wild-type lifespan. It is also essential that a new method has the resolution to accurately quantify changes between test conditions and the statistical power to detect significant changes. In our previous study, we discovered that the Myc family of transcription factors are determinants of C. elegans longevity. mml-1 and mxl-2 encode the C. elegans homologs of mammalian Mondo A/Carbohydrate Response element binding protein (ChREBP) and Mlx, respectively. In both C. elegans and mammals, these Myc-family members heterodimerize to regulate transcription. We found that loss of either mml-1 or mxl-2 significantly decreases normal lifespan, as measured by either a traditional lifespan assay or by Replica Set (Figure 4A-B, yellow and maroon). In contrast to the MML-1::MXL-2 complex, we found that loss of either mdl-1 (homologous to mammalian Mad) or mxl-1 (Max) significantly increased C. elegans lifespan as measured by either methodology (Figure 4 purple and blue, respectively, in both panels).
A serious limitation to the traditional approach for measuring longevity is throughput. Both methods rely on movement to call whether an animal is alive or dead, which becomes increasingly difficult to assess. Young animals will move throughout a plate in the absence of stimuli and are thus easy to score. Aging C. elegans become increasingly sedentary but will respond to a light touch to the head by a reversal movement on a plate. However, as animals become older the ability to move backward diminishes and becomes increasingly uncoordinated. Ultimately animals become paralyzed, a phenotype strongly resembling sarcopenia, and when scoring via the traditional method viability can only be determined by observing subtle twitch at the extreme anterior tip of the animal. In contrast, when scoring viability via the Replica Set method, the liquid is added to the well, which acts as a stimulus that generates a thrashing response that can be quantified as a readout of healthspan8. Movement in the liquid is easier to observe for even older animals: chronologically age-matched decrepit animals produce subtle head movements on dry plates but a more pronounced (albeit slow) body bend in liquid. Finally, when scoring Replica Set, the whole well is within the field of view (~1.1 cm in diameter) and all animals are in suspension- allowing observation of all animals simultaneously. In contrast, when scoring a 6 cm plate via the traditional method, one must scan across the entire plate -searching through the bacterial lawn and along the edges for animals. The net consequence of these differences is that the throughput when using the Replica Set method is at least an order of magnitude greater than the traditional approach, which makes it possible to simultaneously quantify changes in lifespan across more than 100 conditions in a single experiment with one investigator. For example, from a genome-wide feeding-based RNAi screen we previously identified 159 genes that were necessary for the increased lifespan conferred by decreased daf-2/insulin-like signaling8. In that analysis, we quantified the changes in lifespan in wild-type, a long-lived daf-2(e1370) mutant, and short-lived daf-2(e1370);daf-16(mgDf47) double mutant animals (Figure 5A), which allowed us to decipher the genetic relationships between insulin-like signaling and over 100 progeric gene inactivations. Further, we assessed how these progeric gene inactivations altered healthspan (at the time called &#34;activespan&#34;) by observing the decline in C. elegans thrashing across replicates over time (Figure 5B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57819/57819fig1.jpg" /> 
Figure 1: The advent of feeding based RNAi lead to an era of gene discovery in aging research, yet most gerogenes remain poorly studied. (A) Many gerogenes were initially discovered from large scale functional genomic screens. Of the more than 900 C. elegans gerogenes discovered to date, many were identified using feeding-based RNAi, highlighting the value of functional genomic approaches in gene discovery. The graph shows the number of gerogenes discovered per manuscript using RNAi, based on phenotype annotation (See Table of Materials) for phenotype ontology terms extended life span, shortened life span, and life span variant. See Supplemental File 1 for the full list of studies that discovered gerogenes. (B) Most gerogenes remain poorly studied. In contrast to well-studied gerogenes like daf-16/FOXO (arrow), which has more than 800 references, the majority of gerogenes have fewer than 10 references (general reference- not necessarily focused on lifespan). Reliable high-throughput methods will be essential to derive deeper insight into the genetic inter-relationships between gerogenes. The graph is based on mappings between publications in PubMed and the C.elegans gerogenes discovered from RNAi phenotypes. See Supplemental File 2 for the full list of gerogenes and number of studies associated with each. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/57819fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57819/57819fig2.jpg" /> 
Figure 2: The Traditional and the Replica Set Method for scoring C.elegans lifespan (A). The Traditional Method for scoring C. elegans lifespan. Several small synchronized populations of isogenic animals per condition are followed over time. The same population of animals is followed throughout the study course. Viability is assessed by movement, which may be stimulated by gentle prodding. Animals that fail to move are scored as dead and are removed (aspiration shown) until no viable animals remain. (B). The Replica Set Method for scoring C. elegans lifespan. A large population of age-synchronized isogenic animals are distributed across a number of identical replicate plates. At each time point, a single replicate is scored: a mild buffered solution (M9) is added, which stimulates movement. Animals that fail to move spontaneously after flooding wells are also assessed via touch stimulus. The scoring duration for the experiment is determined prior to the start. Each animal is scored only once and longevity for the larger population is derived from many independent observations. (C). The Replica Set approach is a high throughput method to quantitatively measure C. elegans lifespan. 100 or more independent RNAi clones can be tracked simultaneously. HT115 E. coli expressing dsRNA for a given RNAi clone is shown. Practically, every 24 samples from the 96-well plate are divided into a single 24-well plate. Each resulting 24-well plate has a negative (i.e. empty vector, red well) and positive control (green well) randomly distributed within a collection of RNAi clones (yellow wells). Typically, the first well (A1) in a collection contains an empty vector. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/57819fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57819/57819fig3.jpg" /> 
Figure 3: The graphical user interface (GUI). (A). The main plot window interface showing the default welcome screen. This is what is displayed upon opening the software. Differences in appearance between platforms should be minimal due to use of a platform-independent windowing toolkit. (B). Overview of menu options, for the drop-down menus available from the main plot window. (C). Example plot output for both Replica Set style (left) and traditional Kaplan-Meier style (right) data. The data displayed was collected in independent experiments. Exported plots do not include pre-plotted axis labels, for maximum flexibility in adding such labels. To facilitate this, the axes are always divided into increments of 20% for the Y-axis, and increments of 5 for the X-axis. In this example, axis and line labels (strain/treatment) were added to the saved plots, using a very simple and common image editing tool. (D). An example of output from the &#34;TrialView&#34; functionality, allowing for the visual comparison between results of independent trials for Replica Set style datasets. This plot shows the result between two different trials and the corresponding pooled results for daf-2 EV(RNAi) (blue, closed circles), N2 EV (RNAi) (black, closed circles), and daf-2 with daf-16 (RNAi) (red, open diamonds). TrialView allows for quickly checking for trial-specific data issues that might affect the quality of the fit of the pooled dataset. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/57819fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57819/57819fig4.jpg" /> 
Figure 4: The Traditional and Replica Set methods produce similar results. Loss of either component of the MDL-1(Mad)::MXL-1(Max) heterodimer increases lifespan. In contrast, loss of either component of the MML-1 (Mondo/ChREBP)::MXL-2(Mlx) decreases lifespan This figure is reprinted from reference20 with permission via a Creative Commons Attribution (CC BY) license (See Materials). (A). Kaplan-Meier results with the traditional method. (B). Logit curve fit using the Replica Set method. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/57819fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57819/57819fig5.jpg" /> 
Figure 5: The Replica Set method can decipher genetic interactions based on changes in lifespan (A) and alterations in healthspan (B) for over 100 RNAi clones simultaneously. This figure is reprinted from8 with permission under the Creative Commons Attribution-Non-Commercial 4.0 International License (CC-BY-NC) (See Materials). (A) Genetic lifespan analysis of progeric gene inactivations in the context of decreased insulin-like signaling (ILS) (daf-2, x-axis) and in the absence of daf-16/FoxO (Y-axis), a central transcriptional effector of ILS21. Gene inactivations with similar functions as daf-16 do not further shorten lifespan in the absence of daf-16 (black dots). Gene inactivations with functions completely independent from daf-16 shorten both genetic backgrounds similarly (grey). Gene inactivations where the negative effect on lifespan in daf-2 &#62; daf-2;daf-16, suggests function in parallel (white). (B) Changes in thrashing rates over time can derive the average healthspan for many genetic perturbations (x-axis) while assessing changes in lifespan (y-axis). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/57819fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure S1: Workflows in WormLife. Illustration of the steps of some guided workflows (sometimes termed &#34;wizards&#34;). In each of these cases, after the last step in the workflow, the focus is returned back to the main plot window. (A) The data import workflow for Replica Set style datasets (B) The data import workflow for traditional Kaplan Meier style datasets. (C) The workflow for adding lines to a plot for Replica Set style datasets. (D) The workflow for adding lines to a plot for traditional Kaplan Meier style datasets. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/figure_S1.tiff">Please click here to download this file.</a>
Supplemental File 1: Studies that have identified gerogenes. The advent of feeding-based RNAi led to an era of gene discovery for phenotypes that were not tractable by forward genetics, including aging. Listed, in the order of the number of gerogenes discovered, are independent studies that identified genes whose activity altered lifespan. Note that the study that identified the most gerogenes utilized the replicate set method8. The nature of how gene inactivations altered lifespan is also indicated: longevity and progeric genes are those that increased or decreased lifespan when inactivated, respectively. &#34;Life span variant&#34; refers to cases where directionality of change (i.e. increased or decreased lifespan) was not specified or has not been curated. Data from WormBase WS262 (January 2018) (See Materials), with the addition of RNAi-treatment lifespan results from reference10, which are not yet included in the curated collection of WormBase RNAi phenotypes. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/Supplemental_File_1.xlsx">Please click here to download this file.</a>
Supplemental File 2: The number of studies associated with each gerogene. Most gerogenes are poorly studied. While some genes, like daf-16/FoxO, have been the subject of much research attention, more than 400 gerogenes have fewer than 10 associated publications. Data from WormBase WS262 (January 2018), with the addition of RNAi-treatment lifespan results from10 which are not yet included in the curated collection of WormBase RNAi phenotypes. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/Supplemental_File_2.xlsx">Please click here to download this file.</a>
Supplemental File 3: Preparation of common reagents for C. elegans experiments. (A). Recipe for standard NGM and RNAi plates. (B). Recipe for M9 buffer and hypochlorite solution. (C). Preparation of LB +Amp/Tet plates. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/Supplemental_File_3.xlsx">Please click here to download this file.</a>
Supplemental File 4: Replica Set example data. An example dataset from a Replica Set lifespan experiment. This dataset is already formatted to be suitable for import/analysis. Includes two trials per condition (combination of strain/genotype and RNAi). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/Supplemental_File_4.csv">Please click here to download this file.</a>
Supplemental File 5: Traditional longitudinal example data. An example dataset from a traditional longitudinal lifespan experiment, with right-censoring, formatted for ready import/analysis using Kaplan-Meier survival plot functionality. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/Supplemental_File_5.csv">Please click here to download this file.</a>

Watch this Scientific Journal Video about The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan at JoVE.com

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

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.

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

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

the-replica-set-method-high-throughput-approach-to-quantitatively

Nanyang Technological University

Research

JoVE Journal

Genetics

8.9K Views.  University of Rochester Medical Center. Hier beschrijven we de replicaset methode, een benadering tot kwantitatief maatregel C. elegans levensduur/overleving en de healthspan in een high-throughput en robuuste manier, waardoor screening van vele voorwaarden zonder in te boeten op kwaliteit van de gegevens. Dit protocol details van de strategie en biedt een software-instrument voor de analyse van gegevens van de replicaset.

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

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Biochemistry

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Biology

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

High-Throughput Assay to Measure Caenorhabditis elegans Food Intake and Lifespan

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

Behavior

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Unveiling Aging Mysteries: Lifespan Machine for High-Throughput Insights

High-Throughput Behavioral Aging and Lifespan Assays Using the Lifespan Machine

Genetically identical animals kept in a constant environment display a wide distribution of lifespans, reflecting a large non-genetic, stochastic aspect to aging conserved across all organisms studied. This stochastic component means that in order to understand aging and identify successful interventions that extend the lifespan or improve health, researchers must monitor large populations of experimental animals simultaneously. Traditional manual death scoring limits the throughput and scale required for large-scale hypothesis testing, leading to the development of automated methods for high-throughput lifespan assays. The Lifespan Machine (LSM) is a high-throughput imaging platform that combines modified flatbed scanners with custom image processing and data validation software for the life-long tracking of nematodes. The platform constitutes a major technical advance by generating highly temporally resolved lifespan data from large populations of animals at an unprecedented scale and at a statistical precision and accuracy equal to manual assays performed by experienced researchers. Recently, the LSM has been further developed to quantify the behavioral and morphological changes observed during aging and relate them to lifespan. Here, we describe how to plan, run, and analyze an automated lifespan experiment using the LSM. We further highlight the critical steps required for the successful collection of behavioral data and high-quality survival curves.

Author Spotlight: Automated Lifespan Monitoring &#8211; Discovering Aging Dynamics with the Lifespan Machine 

The imaging platform "The Lifespan Machine" automates the lifelong observation of large populations. We show the steps required to perform lifespan, stress resistance, pathogenesis, and behavioral aging assays. The quality and scope of the data allow researchers to study interventions in aging despite the presence of biological and environmental variation.

Genetically identical animals kept in a constant environment display a wide distribution of lifespans, reflecting a large non-genetic, stochastic aspect ...

Lifespan Analysis: Measuring C. elegans Longevity

Source: Cornwell, A. B., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57819/the-replica-set-method-high-throughput-approach-to-quantitatively">The Replica Set Method: A High-throughput Approach to Quantitatively Measure&#160;Caenorhabditis elegans&#160;Lifespan</a>.&#160;J. Vis. Exp.&#160; (2018).
This video describes a traditional, plate-based method of measuring the lifespan of C. elegans. The example protocol measures the lifespan of worms treated with RNAi.

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

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