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).

Figure 1 depicts a schematic of lipid feeding using different experimental settings.
1. Preparation of lipid-conditioned bacteria
<ol>
	<li>Prepare the Bacterial dilution dietary restriction (BDR) base solution by dissolving 5.85 g of NaCl, 1.0 g of K2HPO4, and 6.0 g of KH2PO4 (see Table of Materials) in 999 mL of deionized water. Adjust the pH to 6.0 with 0.5 M KOH, and then filter th...

<ol>
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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#34; 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>lipl-4Tg</td>
			<td>MCW Lab</td>
			<td>N/A</td>
			<td>Transgenic C. elegans</td>
		</tr>
		<tr>
			<td>lipl-4Tg;fat-3(wa22)</td>
			<td>MCW Lab</td>
			<td>N/A</td>
			<td>Transgenic C. elegans</td>
		</tr>
		<tr>
			<td>Luria Broth Base</td>
			<td>ThermoFisher</td>
			<td>12795-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</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 C. elegans food</td>
		</tr>
		<tr>
			<td>Potasium phosphate dibasic trihydrate (K2HPO4&#183;3H2O)</td>
			<td>Sigma</td>
			<td>P5504-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Potasium phosphate monobasic (KH2PO4)</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&#160;</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 (Na2HPO4&#183;7H2O)</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</sup...

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 ...

This protocol describes two lipid supplementation strategies with a combination of longitudinal and transcriptional analysis in the model organism, C.elegans. The technique describes how to examine transcriptional changes using few worms or dissected worm tissues and how the liquid feeding for a bug population could be coupled with hydro techniques. Individuals performing this technique must practice tissue dissection because of the short time window to perform the experiment to achieve adequate transcriptional analysis.To begin, collect OP50 bacteria by centrifuging the overnight-grown culture at 4, 000 G for 10 minutes at room temperature. Discard the supernatant. Suspend the bacterial pellet in 20 milliliters of bacterial dilution, dietary restriction or BDR base by vortexing and again, repeat the centrifugation for 10 minutes.After discarding the supernatant, re-suspend the bacterial pellet in the BDR medium to prepare a 20 X BDR bacterial stock. Dilute the bacterial stock to 5X using a BDR medium before use. Using ethanol or dimethyl sulphoxide as the solvent, prepare a lipid stock solution in a new autoclaved glass vial and fill the glass vial with argon or nitrogen to prevent oxidation.Transfer the lipid stock solution to the BDR bacteria solution to achieve the desired final concentration in feeding conditions. Mix it thoroughly by vortexing for 20 seconds. Prepare the vehicle controlled by mixing the same volumes of filtered ethanol or dimethyl sulphoxide with BDR bacteria.Perform lipid supplementation and liquid culture by transferring the desired amount of lipid or vehicle control to each well of a 12 well plate with three to four replicates for each feeding condition. Mix the synchronized Caenorrhabditis elegans worm suspension with 5X BDR bacteria in a one-to-one ratio to achieve a final concentration of 1, 500 worms per milliliter and 2.5X for bacteria. Then transfer two milliliters of the worm bacteria mixture to each well of the 12 well plate.When done wrap the 12 well plate with foil and shake the plate in a 20 degree Celsius incubator at 100 RPMM for the desired incubation length. For lipid supplementation on plates, seed one milliliter of the lipid conditioned bacteria onto the center of a 10 centimeter nematode growth medium or NGM agar plate. Ensure that the final working solution is vortexed multiple times between seeding the two plates when working with a high number of plates.Dry the plate in the dark inside a biosafety hood. Transfer 3000 worms from the synchronized worm culture to the 10 centimeter plate. Dry the plate in a biosafety hood until worms that were swimming on the lipid supplemented plates, start crawling.Once dried, incubate the lipid conditioned plate in a 20 degree Celsius incubator for the desired length of time and if using polyunsaturated lipids protect the plate from light. Prepare 20 microliters of final lysis solution for each sample by mixing 0.2 microliters of DNase I with 19.8 microliters of lysis solution in a PCR tube and mix it well by pipetting up and down five times. To extract the RNA from the small number of whole worms, remove the bacteria by transferring 15 to 20 worms from the bacterial lawn to a freshly unseeded NGM plate.Transfer 15 to 20 worms from the unseeded NGM plate to the PCR tube containing 20 microliters of the freshly prepared final lysis solution with the minimum number of bacteria and incubate the lysis reaction for five minutes at room temperature. Once the incubation is over, keep the sample in a nice cold water bath and probe sonicate the worms four times, five seconds each time with 30%amplitude and pulse for 15 seconds between each time of sonication. Incubate the tube at room temperature for five minutes before adding two microliters of stop solution into the lysis reaction and mix it by gentle tapping.Incubate the reaction for two minutes at room temperature and then set the tube on ice. To extract RNA from the worm tissue, pick around 20 worms from the bacterial lawn and place them into a fresh unseeded NGM plate to remove the bacteria from the worms. Transfer 20 worms with as few bacteria as possible to a watch glass containing 500 microliters of M9 solution containing four micromolar levamisole.When the worms are immobilized, dissect the germline or intestine using a 25 gauge needle that can be attached to the one milliliter syringe. Using an autoclaved glass pipette, transfer the dissected tissues into the PCR tube and settle the tube on ice for two minutes allowing the deposition of the material at the bottom. Remove the supernatant from the PCR tube before adding 20 microliters of final lysis solution and mix it well by tapping.After incubating the lysis reaction for five minutes, add two microliters of stop solution and tap the tube multiple times. Incubate the tube for two minutes before proceeding to the reverse transcription. In the validation study, RNA extracted from a large population and a few worms showed similar induction of the neuropeptide processing genes, egl-3 and egl-21.It suggests that the RNA extraction from a few worms can be a valid alternative to standard CDNA synthesis techniques from large populations. In the dissected intestine of the lipl-4 transgene worms, supplemented with dihomo-gamma-linolenic acid, or DGLA, the neuronal neuropeptide processing genes were not induced. DGLA supplementation at different concentrations rescued lifespan extension in the worms upon fat-3 knockdown.Worms with lipid supplementation may also be processed for RNA sequencing, proteomics, metabolomics and behavioral studies to identify additional phenotypes under these specific conditions. This methodology can be applied to aging research, lipid biology and any other research area that aims to establish a relationship between a certain phenotype and a nutritional factor or metabolite.

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1.3K visualizações.  Baylor College of Medicine. Este protocolo descreve duas estratégias de suplementação lipídica com uma combinação de análise longitudinal e transcricional no organismo modelo, C.elegans. A técnica descreve como examinar as mudanças transcricionais usando poucos vermes ou tecidos de vermes dissecados e como a alimentação líquida de uma população de insetos poderia ser acoplada a técnicas hidráulicas. Os indivíduos que realizam essa técnica devem praticar a dissecção tecidual devido à curta janela de ...