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.

1. Traditional Method for Scoring C. elegans Longevity
<ol>
	<li>Preparation of reagents
	<ol>
		<li>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, wh...

<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 animal...

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 border="0" cellpadding="0" cellspacing="0" width="1120">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">IPTG (isopropyl beta-D-1-thigalactopyranoside)</td>
			<td style="width:108px;">Gold Bio</td>
			<td style="width:119px;">12481C100</td>
			<td style="width:527px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">FuDR (5-Fluoro-2&#39;-deoxyuridine)</td>
			<td style="width:108px;">Alfa Aesar</td>
			<td>L16497</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:367px;">24 Well Culture Plates</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td style="width:119px;">#662102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Retangular non-treated single-well plate, 128x86mm</td>
			<td style="width:108px;">Thermo-Fisher</td>
			<td style="width:119px;">242811</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">600 &#181;L 96-well plates</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td style="width:119px;">#786261</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">2mL 96-well plates</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td style="width:119px;">#780286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Air-permeable plate seal</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">60941-086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">96-pin plate replicator</td>
			<td style="width:108px;">Nunc</td>
			<td style="width:119px;">250520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">bacto-peptone</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">90000-368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">bacteriological agar</td>
			<td style="width:108px;">Affymetrix/USB</td>
			<td style="width:119px;">10906</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:367px;">C. elegans RNAi clone library in HT115 bacteria- Ahringer</td>
			<td style="width:108px;">Source Bioscience</td>
			<td style="width:119px;">C. elegans RNAi Collection (Ahringer)</td>
			<td>See also Kamath et. al, Nature 2003.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:367px;">C. elegans RNAi clone library in HT115 bacteria- Vidal</td>
			<td style="width:108px;">Source Bioscience</td>
			<td style="width:119px;">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 height="21">
			<td height="21" style="height:21px;width:367px;">WormLife- Software for Replica Set Survival Analysis</td>
			<td style="width:108px;">Samuelson Lab</td>
			<td style="width:119px;">N/A</td>
			<td>https://github.com/samuelsonlab-urmc/wormlife</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L4440 Empty Vector Plasmid</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">1654</td>
			<td>https://www.addgene.org/1654/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Wormbase</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.wormbase.org/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">OASIS</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>https://sbi.postech.ac.kr/oasis2/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Graphpad Prism</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>https://www.graphpad.com/scientific-software/prism/&#160;</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 ...

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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 ...

Hundreds of genes have been identified whose function affects how an organism ages. But many of their relationships are poorly understood. This method can help answer a key question in the aging field.Namely, what are the genetic inter-relationships between gerA genes? The main advantage of this technique is an increase in throughput, the number of genetic predebations that can be quantitatively measured simultaneously is increased by at least an order of magnitude. Lifespan data, collected with this procedure can be plotted and analyzed using software we provide with this publication.In addition to provide insight into lifespan, this method can also be applied to other age-related phenotypes, such as stress resistance and changes in health plan. For this procedure, create a fresh stamp of the RNAi sub-library. First, aliquot each well of a 96-well plate, with 200 microliters of LB with ampicillin, using a 12-channel pipetter.Sterilize a 96-pin plate replicator by sequentially immersing the pins in 50%bleach followed by ultra pure water. Then dip the tips in ethanol, and carefully flame the tips. Next, carefully remove adhesive foil cover of a frozen, 96-well glycerol stock library plate, and gently but firmly grind the tips of the sterilized plate replicator into the wells of the still frozen glycerol stocks.Then inoculate the cultures and seal the inoculated plate with the permeable membrane. Allow cultures to grow overnight on the bench top. The next day, sterilize the plate replicator, and carefully remove the seal from the overnight culture, and immerse tips of the replicator pins.Next, inoculate a rectangular LB ampicillin and tetracycline agar plate with the pins. Apply even pressure, and use a small, circular motion to transfer the bacteria without letting the spots get too crowded. Then, incubate the plates overnight at 37 degrees Celsius.The next day, for every 10 replica plates needed, load each well of a deep-welled 96-well plate with 1.5 milliliters of LB with ampicillin. Next, inoculate the wells with the cultured bacteria. Then, seal the plate with a breathable membrane, culture it for 20 hours with shaking.Control wells should be scattered across the 96 wells, such that when the 96-well sub-library plate is divided into groups of 24, each division of 24 wells has one of each of the controls. The following day, load 120 microliter aliquots of the culture, to the 24-well plates, using a six-well multichannel pipette, with adjustable tip spacing. Then, dry plates uncovered in a laminar flow hood until all liquid has been absorbed.Do not let them over dry. Be sure to maintain your focus when aliquoting liquid cultures, to ensure that the proper bacterial culture is put into the proper well in the 24-well plate. Replica set experiments start with the set of independent, 24-well replicate plates, each seeded with the same bacteria RNAi clones.For lifespan experiments, 15 to 20 L1 animals are added to each plate well, and allowed to grow to the L4 stage. At L4, FUdR is added to all wells across replicates. Later in the same day, a replicate plate can be taken from the set and scored as the first time point.To score lifespan, liquid is added to the wells of the plate to induce animal movement. Animals that do not start to move are prodded gently with a worm pick. The total number of animals, and the number of dead animals is then recorded.The plate from the first observation is then discarded. At the next observation time point, another replicate plate is taken from the set, and scored in a similar fashion. This is repeated at the determined scoring interval.Each time with a new plate from the replicate set. Continue scoring a set of plates until no animals remain alive. First, synchronize a population of animals with the same bleach-based prep method used for the traditional lifespan assay.Then, using a six-well adjustable spacing pipette, and reagent reservoir, seed 15 to 20 L1 animals into each well of the prepared 24-well plates. While seeding the plates, periodically mix the L1 animals into solution to maintain even distribution. Now, let the animals develop to L4.The timing of L4 development must be known for this protocol.Periodically monitor the development of the worms. When animals reach L4, add 24 microliters of 50x FUdR to each well, and return the plates to the incubator. Using L4 animals is critical.If hermaphrodites reach reproductive maturity before adding FUdR, their progeny will survive and eat the food. Conversely, adding FUdR too early leads to ruptured animals. At the desired time points, use one replicate plate to score viability.To do so, flood the well with M9 solution. Before scoring viability each day, sensor any wells that do not have food, appear contaminated, or have animals with morphological or developmental defects. For each well, record the total animals per well, and check the status of non-moving worms, by gently touching each on the head with a worm pick.After counting all the non-moving dead in a plate, discard the plate. For this procedure, grow transformed e. Coli overnight in LB with ampicillin.The next day, concentrate the bacteria by centrifugation at 3, 000 G's for 15 to 20 minutes. Resuspend the bacteria in 1/10 of the starting volume. Next, aliquot 200 microliters of the 10x bacteria on to six-centimeter plates.Prepare three or four plates per test condition, and include two extra plates in case of contamination. Dry the plates in a laminar flow batch, until all the liquid has been absorbed or evaporated. Once dried, store the plates in a plastic box overnight at room temperature to induce the double-stranded RNA production.Then, store the plates for up to two weeks at four degrees Celsius. For the animals, collect gravid, adult animals in M9.Wash them twice with M9.Repeating the spin at aspiration. Then, resuspend C.elegans in four milliliters of M9, and add two milliliters of hypochlorite solution.Now, immediately vortex the worms for three minutes, with periodic, vigorous shaking. Then, under a dissecting microscope, look for a cloud of eggs. If the cloud isn't seen, vortex the worms for an additional 10 to 20 seconds.Next, quickly wash the eggs with M9, two times, as before. And resuspend the washed eggs in three milliliters M9.Then, transfer the eggs to a new, 15-milliliter tube, and incubate tube at 20 degrees Celsius with gentle rotation, and allow the embryos to hatch overnight. The next day, determine the density of the prepared L1 solution by calculating the average number of L1s per microliter in three, 10-microliter drops.Load the prepared bacteria seeded plates with 50 L1 animals, each. Set up the plates in a 20 degree Celsuis incubator, until the embryos develop to the L4 stage. Normal development from L1 to L4 takes about 40 hours.Then, to prevent progeny production, add 160 microliters of 50x FUdR to each plate. Then remove the males, as lifespan is typically measured only with hermaphrodites. Once completed, repack the plates into the 20 degrees Celsuis incubator.Until all the worms are dead, score their viability on a daily basis. To do so, gently touch each non-moving nematode on the head with a platinum wire or eyelash. Non-reactive animals are dead, and must be removed from the plate.Take note of any anomalies during this routine. Replica set and traditional methods for assaying C.elegans lifespan produce similar results with wild-type N2 animals. For example, feeding-based RNAi knockdown of mml-1 or mxl-2 significantly decreased normal lifespans.This was measured by both a traditional lifespan assay, and by the replica set assay. Extended longevity can also be detected. Seen here, RNAi for mdl-1 or mxl-1 significantly increased the lifespans of C.Elegans when measured by either methodology.Using the replica set method, it is possible to simultaneously quantify changes in lifespans across more than 100 conditions, which would not be reasonable with a more traditional approach. For example, a genome-wide feeding-based RNAi screen identified 159 genes necessary for the increased lifespan conferred by decrease daf-2 insulin-like signaling. The relationship between insulin-like signaling and these genes was further described by measuring the health span of the animals.Health was indicated by c. Elegans thrashing when suspended in solution. After watching this video, you should have a good understanding of how to use feeding-based RNAi to measure lifespan of C.Elegans.But RNAi is versatile and can be applied for many different phenotypes. Once mastered, the replica set method allows lifespan to be assessed for over 100 RNAi clones each day, if it is performed properly. Early and late time points can be scored quickly.All the animals in a well will be either alive and moving, or dead. Scoring takes longest when animals are starting to die. Stagger the set up of independent trials with this in mind.

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8.4K Views.  University of Rochester Medical Center. Hundreds of genes have been identified whose function affects how an organism ages. But many of their relationships are poorly understood. This method can help answer a key question in the aging field.Namely, what are the genetic inter-relationships between gerA genes? The main advantage of this technique is an increase in throughput, the number of genetic predebations that can be quantitatively measured simultaneously is increased by at least an order of magnitude. Lifespan data, coll...