We thank P. Svay for maintenance support. This work was supported by NIH grants R01AG045183 (MCW), R01AT009050 (MCW), R01AG062257 (MCW), DP1DK113644 (MCW), March of Dimes Foundation (MCW), Welch Foundation (MCW), HHMI investigator (M.C.W.), and NIH T32 ES027801 pre-doctoral student fellow (M.S.). Some strains were provided by the CGC, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

<ol>
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The authors have no conflicts of interest.

Lipid supplementation has been employed in aging research to elucidate the direct impact of certain lipid species on healthy aging6,7,23,26,27,31. However, the lipid supplementation procedure can be challenging, and any inconsistency between experiments can cause non-reproducible results. Here, the first detailed step-by-step protocol is documented to guide new scientists to avoid the potential pitfalls caused by technical imprecision. The critical steps in this protocol will be discussed in detail in the following paragraphs. The lipid research toolbox is also expanded by introducing RNA isolation from only a few worms and specific worm tissues after lipid supplementation. When considering the methodology to examine transcript levels, the qRT-PCR with a few worms or dissected tissues is outstanding for analyzing a few transcripts or examining certain tissue-specific transcriptional changes. Moreover, using these methodologies could overcome the step of worm amplification that can take approximately 5-6 extra days. At the same time, lipid feeding followed by bulk RNA extraction is more cost-effective and a valid alternative when a larger set of target genes needs to be analyzed.
Several steps can be critical for the reproducibility of lipid feeding effects. The first aspect is related to the bacterial conditions. It is suggested to use fresh bacterial plates that are not older than 7 days for inoculation. It is recommended to use bacteria prepared in BDR medium within 1 week. Bacteria that have been mixed with lipids have to be used right away. Lipids with bacteria should not be stored even at 4 &#176;C, as the bacteria will metabolize the lipids. The washing steps in the BDR base and resuspension in the BDR medium are critical for bacteria conditions, as bacteria were grown in LB, and fed directly to worms always eliminates the profound effects of lipid supplements. The second factor is associated with worm conditions. C. elegans must be non-starved for at least three generations prior to the bleaching step for egg preparation to ensure that they are in a healthy and stable metabolic state. It is also crucial to culture C. elegans on agar plates prior to lipid supplementation; this includes before and after the synchronization step.
Worms that adapted to liquid cultures for prolonged times are partially starved; starvation elevates the baseline for pro-longevity genes, which leads to a weakened effect of lipid supplementation. If the metabolic drift and changes using arrested L1 larvae are concerns, a valid alternative would be to directly plate the eggs. When only a few worms are needed to perform lifespan or gene expression analysis, it is possible to plate eggs directly on lipid-conditioned plates and re-synchronize them by hand-picking at the L4 stage for subsequent experiments. However, if large amounts of worms are needed when hand-picking L4 is not applicable, plating eggs directly is not ideal. Eggs hatching after bleaching from gravid adults can occur at different time points and cause the population to be unsynchronized, which would interfere with the transcriptional analysis. The third critical part is linked to the lipid storage conditions; when supplementing PUFAs, extra attention is needed as these molecules are sensitive to light and prone to oxidization in the air.
Multiple lipid-feeding conditions, including worm stages, supplementation length, and concentrations, require further investigation when testing new lipid molecules. L4, day-1 adult, and day-2 adult worms are usually the starting point for testing at different worm stages. Notably, when feeding L4 worms, if the incubation time ends around the nematode molting phase, a large variation is expected, which greatly influences the significance and reproducibility of the results. An additional challenge for using day-1 or day-2 adult worms is related to the progenies that can complicate the gene expression analysis. In this instance, RNA extraction from a few whole worms is more reliable than bulk populations. Different lipid molecules have different concentration ranges to produce physiological effects; thus, a series of concentrations from 1 &#181;M to 1 mM is suggested to be tested.
There are a few limitations to be considered when choosing the feeding method. First, when lipids cannot be absorbed or ingested by the worms, it is challenging to use a supplementation method to test their biological effect in C. elegans. With current technologies, mass spectrometry or SRS coupled with 13C- or 2H-labeled lipid compounds39 are valid tools to test lipid uptake into the worm body. Second, these feeding methods are not optimized for high-throughput investigative techniques. For lipid supplementation with bulk worms, sample preparation from the liquid feeding method is faster than on-plate feeding, because the liquid cultures can be directly transferred to microcentrifuge tubes instead of washing off from the feeding plates. To ensure that the RNA extracted is at the state of harvest, it is suggested not to let more than 15-20 min pass between the point of setting the worms on ice to grinding them in the RNA extraction solution. It is recommended to process fewer conditions every 15 min when a high number of samples needs to be processed. For whole worm RNA extraction from a few animals, the hand-picking step is the rate-limiting step, while for dissecting tissues, it is crucial to act in a time-efficient manner to avoid long-term exposure to a non-physiological environment. Similar to bulk RNA extraction, picking worms or dissecting tissue samples within 10 min is preferable.
Despite the limitations, these supplementation methods can be used beyond lipid research to aid the identification of any nutritional and medicinal effects. The procedures reported here are not only limited to aging research but alternative phenotypes&#160;to assess organelle fitness and cell metabolic homeostasis. The supplementation method with bulk population can be coupled with RNA-seq for transcriptome analysis, mass spectrometry for metabolomic and proteomic analysis, or Western blot for analysis of specific protein markers, while the lipid supplementation with a few worms can be combined with imaging and behavioral analysis.

Lipids 
Lipids are small hydrophobic or amphipathic molecules soluble in organic solvents but insoluble in water1,2. Distinct lipid molecules differentiate from each other based on the number of carbons contained in their chains, location, number of double bonds, and bound structures, including glycerol or phosphates. Lipids play crucial roles within and across distinct cells to regulate organismal functions, including constituting membrane bilayers, providing energy storage, and acting as signaling molecules3,4.
First, lipids are structural components of biological membranes, including the plasma membrane and intracellular subcellular membranes that divide the internal compartments from the extracellular environment. Second, lipids are the major form of energy storage in vertebrate and invertebrate animals. Neutral lipids, including triacylglycerols, are stored for an extended period in various tissues, including in adipose tissue. In the nematode Caenorhabditis elegans, the intestine is the major metabolic fat storage organ; its function is not only involved in digestion and absorption of nutrients, but also in the process of detoxification, which resembles the activity of mammalian hepatocytes. Other fat storage tissues include the germline, in which lipids are essential for developing oocytes, and the hypodermis, which is composed of skin-like epidermal cells3,5. Third, in recent years, more evidence has suggested that lipids are powerful signaling molecules involved in intra- and extracellular signaling by directly acting on a variety of receptors, including G protein-coupled and nuclear receptors, or indirectly via membrane fluidity modulation or post-translational modifications6,7,8,9. Further studies will continue to elucidate the underlying molecular mechanisms of lipid signaling in promoting longevity and healthspan.
Model organisms are important to address specific biological questions that are too complex to study in humans. For example, the roundworm C. elegans is an excellent model for conducting genetic analysis to dissect biological processes relevant to human nutrition and disease10. The highly conserved molecular pathways relevant to human physiology, complex tissues, behavioral patterns, and abundant genetic manipulation tools make C. elegans a remarkable model organism11. For instance, C. elegans is excellent in forwarding genetic screens to identify phenotype-specific genes, as well as in genome-wide reverse genetic screens via RNA interference12.
In laboratories, the nematodes are grown on agar Petri plates seeded with a lawn of Escherichia coli bacteria, providing macronutrients such as proteins, carbohydrates, and saturated and unsaturated fatty acids as sources of energy and building blocks, and micronutrients such as co-factors and vitamins13. Similar to mammals, nematodes synthesize fatty acid molecules from both palmitic acid and stearic acid (saturated 16-carbon and 18-carbon molecules, respectively) that are sequentially desaturated and elongated to a variety of mono-unsaturated fatty acids (MUFAs) and poly-unsaturated fatty acids (PUFAs)14,15,16,17,18. Interestingly, C. elegans is capable of de novo synthesis of all the required fatty acids and core enzymes involved in fatty acid biosynthesis, desaturation, and elongation, facilitating the synthesis of long-chain PUFAs19. Different from other animal species, C. elegans can convert 18-carbon and 20-carbon &#969;-6 fatty acids into &#969;-3 fatty acids with its own &#969;-3 desaturase enzymes. Additionally, worms possess a &#916;12 desaturase that catalyzes the formation of linoleic acid (LA) from oleic acid (OA, 18:1)20,21. Most animals or plants lack both &#916;12 and &#969;-3 desaturases and thus rely on dietary intake of &#969;-6 and &#969;-3 to obtain their PUFAs, whereas C. elegans does not require dietary fatty acids22. Isolated mutants lacking functional desaturase enzymes have been used to study the functions of specific fatty acids in distinct biological processes, including reproduction, growth, longevity, and neurotransmission. The effect of individual fatty acids on specific biological pathways can be addressed using both a genetic approach and diet supplementation16,17,23. To date, lipid research has focused on characterizing genes involved in the lipid synthesis, degradation, storage, and breakdown in neurological and developmental conditions24. However, the roles of lipids in longevity regulation are just beginning to be revealed.
Lipid signaling in longevity regulation 
Lipids play crucial roles in longevity regulation by activating cellular signaling cascades in distinct tissues and cell types. Recent studies have highlighted the active roles of lipids in modulating transcription and cell-cell communication via lipid-binding proteins or recognition of membrane receptors25. Additionally, dietary lipid supplementation offers an excellent tool to dissect how lipid metabolism influences lifespan in C. elegans. Distinct MUFAs and PUFAs have been shown to promote longevity by activating transcription factors26,27.
Longevity models, including the insulin/IGF-1 signaling and the ablation of germline precursor cells, are associated with the MUFA biosynthesis pathway, and MUFA supplementation, including oleic acid, palmitoleic acid, and cis-vaccenic, is sufficient to extend C. elegans lifespan26. Although the longevity effect conferred by the MUFA administration requires further investigation, the underlying mechanism is likely to be mediated by the SKN-1/Nrf2 transcription factor, which is a key activator of oxidative stress response and longevity regulation28,29. Among MUFAs, a particular class of fatty acyl ethanolamides called N-acylethanolamines (NAEs) plays crucial roles in distinct mechanisms including inflammation, allergies, learning, memory, and energy metabolism30. Particularly, the lipid molecule known as oleoylethanolamide (OEA) has been identified as a positive regulator of longevity by promoting the translocation of the lipid-binding protein 8 (LBP-8) into the nucleus to activate the nuclear hormone receptors NHR-49 and NHR-807. Supplementation of the OEA analog KDS-5104 is sufficient to extend lifespan, and induces the expression of genes involved in oxidative stress responses and mitochondrial &#946;-oxidation7,8.
At the same time, the role of PUFAs has also been linked to longevity regulation. Administration of PUFA &#969;-3 fatty acid &#945;-linolenic acid (ALA) promotes longevity by activating the NHR-49/PPAR&#945;, SKN-1/NRF transcription factors and inducing mitochondrial &#946;-oxidation31. Interestingly, peroxidated products of ALA, referred to as oxylipins, activate SKN-1/NRF, suggesting that both PUFAs and their oxidative derivatives can confer longevity benefits23. Supplementation of &#969;-6 fatty acid arachidonic acid (AA) and dihomo-&#947;-linolenic acid (DGLA) extends lifespan via autophagy activation, promoting protein quality control and resulting in the degradation of wasted and toxic protein aggregates27,32. More recently, a cell-non-autonomous signaling regulation mediated by the lipid-binding protein 3 (LBP-3) and DGLA has been shown to be crucial to promoting longevity by sending peripheral signals to neurons, suggesting a long-range role of lipid molecules in inter-tissue communication at systemic levels33. The present study reports each step to perform lipid supplementation with bacteria seeded on plates or bacterial suspension in liquid culture. These methodologies are used to assess lifespan and transcriptional analysis, employing whole body content or dissected tissues derived from a few worms. The following techniques can be adapted to a variety of nutritional studies and offer a valid tool to dissect how lipid metabolism influences longevity and healthy aging.

<table><tbody><tr><td>1.5 mL Pestle</td><td>Genesee Scientific</td><td>93-165P15</td><td>For worm grinding with Trizol</td></tr><tr><td>Agarose</td><td>Sigma</td><td>A9639-500G</td><td></td></tr><tr><td>AmfiRivert cDNA Synthesis Platinum Master Mix</td><td>GenDEPOT</td><td>R5600</td><td>For reverse transcription from bulk worm samples</td></tr><tr><td>Applied Biosystems QuanStudio 3 Real-Time PCR</td><td>ThermoFisher</td><td>A28567</td><td>For qRT-PCR</td></tr><tr><td>Benchmark Scientific StripSpin 12 Microcentrifuge</td><td>Benchmark Scientific</td><td>C1248</td><td>For spin down PCR tubes</td></tr><tr><td>Branson 450 Digital Sonifier, w/ 1/8&quot; tip</td><td>Branson Ultrasonic Corporation</td><td>100-132-888R</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>C298-500</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma</td><td>C8503-25G</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D8418-100ML</td><td></td></tr><tr><td>Eppendorf 5424 R centrifuge</td><td>Eppendorf</td><td>22620444R</td><td>For RNA extraction</td></tr><tr><td>Eppendorf vapo protect mastercycler pro</td><td>Eppendorf</td><td>950030010</td><td>For reverse transcription</td></tr><tr><td>Ethanol, Absolute (200 Proof)</td><td>Fisher Scientific</td><td>BP2818-500</td><td></td></tr><tr><td>Greiner Bio-One CELLSTAR, 12 W Plate</td><td>Neta Scientific</td><td>665180</td><td>12-well plates for licuid feeding</td></tr><tr><td>Greiner Bio-One Petri Dish, Ps, 100 x 20 mm</td><td>Neta Scientific</td><td>664161</td><td>For bacterial LB plates and worm 10-cm NGM plates</td></tr><tr><td>Greiner Bio-One Petri Dish, Ps, 60 x 15 mm</td><td>Neta Scientific</td><td>628161</td><td>For worm6-cm NGM plates</td></tr><tr><td>Invitrogen nuclease-free water</td><td>ThermoFisher</td><td>AM9937</td><td></td></tr><tr><td>Isoproanol</td><td>Sigma</td><td>PX1835-2</td><td></td></tr><tr><td>Levamisole hydrochloride</td><td>VWR</td><td>SPCML1054</td><td></td></tr><tr><td>&lt;em&gt;lipl-4Tg&lt;/em&gt;</td><td>MCW Lab</td><td>N/A</td><td>Transgenic &lt;em&gt;C. elegans&lt;/em&gt;</td></tr><tr><td>&lt;em&gt;lipl-4Tg;fat-3(wa22)&lt;/em&gt;</td><td>MCW Lab</td><td>N/A</td><td>Transgenic &lt;em&gt;C. elegans&lt;/em&gt;</td></tr><tr><td>Luria Broth Base</td><td>ThermoFisher</td><td>12795-084</td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>M2643-500G</td><td></td></tr><tr><td>MicroAmp EnduraPlate Optical 96-Well Fast Clear Reaction Plate with Barcode</td><td>ThermoFisher</td><td>4483354</td><td>96-well qPCR plate</td></tr><tr><td>MicroAmp Optical Adhesive Film</td><td>Applied BioSystem</td><td>4311971</td><td>For sealing the 96-well qPCR plate</td></tr><tr><td>Milli-Q Advantage A10 Water Purification System</td><td>Sigma</td><td>Z00Q0V0WW</td><td>Deionized water used to make all reagents, including buffer and cultural media, unless specified as nuclease-free water in the protocol</td></tr><tr><td>N2</td><td>Caenorhabditis Genetics Center</td><td>N/A</td><td>C. elegans wild isolate</td></tr><tr><td>NanoDrop ND-1000 Spectrophotometer</td><td>ThermoFisher</td><td>N/A</td><td>For measuring RNA concentration</td></tr><tr><td>OP50</td><td>Caenorhabditis Genetics Center</td><td>N/A</td><td>Bacteria used as &lt;em&gt;C. elegans&lt;/em&gt; food</td></tr><tr><td>Potasium phosphate dibasic trihydrate (K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;3H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma</td><td>P5504-1KG</td><td></td></tr><tr><td>Potasium phosphate monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>P0662-2.5KG</td><td></td></tr><tr><td>Power SYBR Green cells-to-Ct kit</td><td>ThermoFisher</td><td>4402953</td><td>For reverse transcription and qPCR from a few worms or worm tissue</td></tr><tr><td>Power SYBR Green Master Mix</td><td>ThermoFisher</td><td>4367659</td><td>For qPCR from bulk worm samples</td></tr><tr><td>Pure Bright germicidal ultra bleach&amp;nbsp;</td><td>KIK International LLC.</td><td>59647210143</td><td>6% house bleach For worm egg preparation</td></tr><tr><td>Pyrex spot plate with nine depressions</td><td>Sigma</td><td>CLS722085-18EA</td><td>Watch glass for dissecting the worms</td></tr><tr><td>RNaseZap RNase Decontamination Solution</td><td>ThermoFisher</td><td>AM9780</td><td></td></tr><tr><td>Sodium cloride (NaCl)</td><td>Sigma</td><td>S7653-1KG</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma</td><td>SX0590-3</td><td></td></tr><tr><td>Sodium phosphate dibasic heptahydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma</td><td>S9390-1KG</td><td></td></tr><tr><td>Thermo Sorvall Legend Mach 1.6R Centrifuge</td><td>Thermo</td><td>7500-4337</td><td>For bacteria collection</td></tr><tr><td>Thermo Sorvall ST 8 centrifuge</td><td>Thermo</td><td>7500-7200</td><td>For worm egg preparation</td></tr><tr><td>TRIzol Reagent</td><td>TheroFisher</td><td>15596018</td><td>RNA extraction reagent</td></tr><tr><td>Turbo DNA-free kit</td><td>ThermoFisher</td><td>AM1907</td><td>For removing DNA contamination in RNA extractions</td></tr><tr><td>Vortexer 59</td><td>Denville Scientific INV</td><td>S7030</td><td></td></tr><tr><td>VWR Disposable Pellet Mixers and Cordless Motor</td><td>VWR</td><td>47747-370</td><td>For worm grinding with Trizol</td></tr><tr><td>VWR Kinetic Energy 26 Joules Mini Centrifuge C1413 V-115</td><td>VWR</td><td>N/A</td><td>For worm collection. Discontinued model, a similar one available at VWR with Cat# 76269-064</td></tr><tr><td>Worm picker</td><td>WormStuff</td><td>59-AWP</td><td></td></tr></tbody></table>

lipid supplementation for longevity and gene transcriptional analysis in caenorhabditis elegans

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are crucial in constituting structural components of cell membranes, storing energy, and as signaling molecules. Regulation of lipid metabolism and signaling is essential to activate distinct longevity pathways. The roundworm Caenorhabditis elegans is an excellent and powerful organism to dissect the contribution of lipid metabolism and signaling in longevity regulation. Multiple research studies have described how diet supplementation of specific lipid molecules can extend C. elegans lifespan; however, minor differences in the supplementation conditions can cause reproducibility issues among scientists in different labs. Here, two detailed supplementation methods for C. elegans are reported employing lipid supplementation either with bacteria seeded on plates or bacterial suspension in liquid culture. Also provided herein are the details to perform lifespan assays with lifelong lipid supplementation and qRT-PCR analysis using a whole worm lysate or dissected tissues derived from a few worms. Using a combination of longitudinal studies and transcriptional investigations upon lipid supplementation, the feeding assays provide dependable approaches to dissect how lipids influence longevity and healthy aging. This methodology can also be adapted for various nutritional screening approaches to assess changes in a subset of transcripts using either a small number of dissected tissues or a few animals.

Validation of transcriptional changes using a few whole worms upon lipid supplementation 
To investigate whether the protocol to extract and retrotranscribe RNA into cDNA from a few whole worms is reproducible and comparable with the data from bulk worms, a long-lived worm strain overexpressing the lysosomal acid lipase lipl-4 in the intestine was employed7,8,33,35. The transcriptional induction of the neuropeptide processing genes egl-3 and egl-21 reported in previous studies7,8,33 was validated (Figure 2A,B). This induction indicates that the RNA extraction method from a few animals is a valid alternative to standard cDNA synthesis techniques from bulk worm cultures.
Validation of transcriptional evaluations using dissected worm tissues upon lipid supplementation 
In C. elegans, the synthesis of 20-carbon PUFAs is dependent on the activity of the desaturase FAT-316,17. Previous studies have reported that fat-3 mutants lack 20-carbon PUFAs, including DGLA16. Previously, it was discovered that the loss of &#916;6-desaturase FAT-3 in lipl-4g worms suppresses the transcriptional induction of the neuropeptide processing genes egl-3 and egl-2133. In addition, DGLA supplementation rescues such induction33. The gene encoding egl-21 is expressed in neurons, while egl-3 is detected in both neurons and intestines36,37. To further test whether DGLA supplementation restores the induction of egl-3 and egl-21 in the intestine or in the neurons, the intestine was dissected out and their transcriptional levels were assessed using qRT-PCR analysis described in steps 4.3 and 5.2 of this protocol. DGLA was supplemented in the source food for 12 h at day-1 adulthood. No transcriptional induction of either egl-3 or egl-21 was found in the intestine (Figure 2C), which is consistent with previous findings36,38.
Validation of lifespan assay upon lipid supplementation 
The relationship between 20-carbon PUFAs and the longevity mechanism inactivating fat-3 was previously explored, specifically in the intestine of lipl-4g worms33. It was found that the fat-3 knockdown fully suppresses the lifespan extension conferred by lipl-433. To assess whether DGLA restores the longevity effected mediated by lipl-4, DGLA was supplemented freshly every other day into the source food at day- 1 of adulthood. It was found that upon fat-3 knockdown, the DGLA supplementation rescues the lifespan extension (Figure 2D)33, indicating a successful lipid supplementation procedure coupled with the lifespan assay.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64092/64092fig01.jpg" /> 
Figure 1: Schematic of lipid feeding using different experimental settings. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64092/64092fig01large.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/64092/64092fig02.jpg" /> 
Figure 2: Validation of the neuropeptide processing genes&#39; transcriptional evaluation using a large population of worms, a few worms, and dissected tissues. (A) RNA extracted from a large population of worms shows the induction of the neuropeptide processing genes&#39; egl-3 and egl-21 transcript levelsin lipl-4 Tg animals. Error bars represent &#177;1 SEM. **** p &#60; 0.0001 by two-tailed Student&#39;s t-test. (B) RNA extraction from a few worms confirms the induction of neuropeptide processing genes&#39; egl-3 and egl-21 transcript levels&#160;in lipl-4 Tg worms. Error bars represent &#177;1 SEM. **** p &#60; 0.0001 by two-tailed Student&#39;s t-test. (C) Neuronal neuropeptide processing genes egl-3 and egl-21 are not induced in dissected intestines. Error bars represent &#177;1 SEM. Statistical analysis with two-tailed Student&#39;s t-test. (D) DGLA supplementation at different concentrations, including 10 &#181;m, 100 &#181;m, and 1 mM rescues the lipl-4Tg longevity effect upon fat-3 RNAi. *** p &#60; 0.001 by log-rank test. This figure is adapted from Savini et al.33. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64092/64092fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans at JoVE.com

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

The present protocol describes lipid supplementation methods in liquid and on-plate cultures for Caenorhabditis elegans, coupled with longitudinal studies and gene transcriptional analysis from bulk or a few worms and worm tissues.

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are ...

lipid-supplementation-for-longevity-gene-transcriptional-analysis

Nanyang Technological University

Research

JoVE Journal

Biology

1.4K Views.  Baylor College of Medicine. The present protocol describes lipid supplementation methods in liquid and on-plate cultures for Caenorhabditis elegans, coupled with longitudinal studies and gene transcriptional analysis from bulk or a few worms and worm tissues.

Video: Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

Genetics

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on animal physiology and behavior. Recently, we showed that modulation of longevity in the nematode Caenorhabditis elegans by food abundance is more complex than previously recognized. The responsiveness of the lifespan to changes in food level is determined by specific genes that act by controlling information processing within a neural circuit. Our framework combines genetic analysis, high-throughput quantitative imaging and information theory. Here, we describe how these techniques can be used to characterize any gene that has a physiological relevance to broad-range dietary restriction. Specifically, this workflow is designed to reveal how a gene of interest regulates lifespan under broad-range dietary restriction; then to establish how the expression of the gene varies with food level; and finally, to provide an unbiased quantification of the amount of information conveyed by gene expression about food abundance in the environment. When several genes are examined simultaneously under the context of a neural circuit, this workflow can uncover the coding strategy employed by the circuit.

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Here, we present a framework to relate broad-range dietary restriction to gene expression and lifespan. We describe protocols for broad-range dietary restriction and for quantitative imaging of gene expression under this paradigm. We further outline computational analyses to reveal underlying information processing features of the genetic circuits involved in food-sensing.

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

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

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.

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

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 ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

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Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy