Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

Summary

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.

Abstract

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.

Introduction

Lipids
Lipids are small hydrophobic or amphipathic molecules soluble in organic solvents but insoluble in water^1,2. 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 molecules^3,4.

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 cells^3,5. 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 modifications^6,7,8,9. 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 ω-6 fatty acids into ω-3 fatty acids with its own ω-3 desaturase enzymes. Additionally, worms possess a Δ12 desaturase that catalyzes the formation of linoleic acid (LA) from oleic acid (OA, 18:1)^20,21. Most animals or plants lack both Δ12 and ω-3 desaturases and thus rely on dietary intake of ω-6 and ω-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 supplementation^16,17,23. 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 factors^26,27.

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 regulation^28,29. 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 β-oxidation^7,8.

At the same time, the role of PUFAs has also been linked to longevity regulation. Administration of PUFA ω-3 fatty acid α-linolenic acid (ALA) promotes longevity by activating the NHR-49/PPARα, SKN-1/NRF transcription factors and inducing mitochondrial β-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 ω-6 fatty acid arachidonic acid (AA) and dihomo-γ-linolenic acid (DGLA) extends lifespan via autophagy activation, promoting protein quality control and resulting in the degradation of wasted and toxic protein aggregates^27,32. 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.

Protocol

Figure 1 depicts a schematic of lipid feeding using different experimental settings.

1. Preparation of lipid-conditioned bacteria

1. 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 through a 0.22 µm filter.
   NOTE: The BDR solution can be stored at room temperature for long-term storage.
2. Prepare the BDR medium by adding 10 µL of 5 mg/mL cholesterol (dissolved in 200-proof ethanol) to every 10 mL of BDR base solution.
3. Streak the OP50 bacteria (see Table of Materials) from -80 °C stock to LB-agar plates and incubate at 37 °C overnight.
   NOTE: After the overnight incubation at 37 °C, the OP50 plate can be stored at 4 °C for up to 1 week for steady feeding results.
4. Inoculate the OP50 bacteria from the OP50 LB-agar plate to LB medium and incubate in a 37 °C shaker overnight (16 h).
   NOTE: The LB medium volume needed depends on the experimental settings. It is recommended to use 200 µL of 5x concentrated bacteria seeded onto each of the 6 cm plates and 1 mL of 5x concentrated bacteria for each of the 10 cm plates and each replicate of the liquid feeding conditions. Thus, 1 mL and 5 mL of initial LB inoculation must be prepared for each lipid-feeding culture, respectively.
5. Collect bacteria by centrifugation at 4,000 x g for 10 min at room temperature. Discard the supernatant.
6. Suspend the bacterial pellet in 20 mL of BDR base, and centrifuge at 4,000 x g for 10 min. Discard the supernatant.
   NOTE: This step is critical to wash off the leftover residues of LB in the bacterial pellet.
7. Suspend each bacterial pellet in the BDR medium to make a 20x BDR bacteria stock. Dilute the 20x BDR bacteria stock with BDR medium to 5x before use.
   NOTE: Use a 1/20 volume of BDR medium of the original LB volume to reach a 20x concentration for the bacteria. The 20x bacteria stock can be stored at 4 °C for up to 1 week.
8. Prepare a lipid stock solution, using ethanol or dimethyl sulfoxide (DMSO) as the solvent in a new autoclaved glass vial, and fill the glass vial with argon or nitrogen to prevent oxidation.
   NOTE: Stock solution concentration is usually between 250 mM and 1 mM.
9. Transfer the lipid stock solution to the desired amount of BDR bacteria solution to achieve the desired final concentration in feeding conditions. Mix thoroughly by vortexing for 20 s.
   NOTE: The final concentration of the lipids in feeding is usually 1 µM to 1 mM, depending on the lipid and testing conditions. It is recommended to run a pilot experiment first to test different concentrations ranging from 0.1 µM to 1 mM if working with new lipids. Mix the lipid stock solution with BDR bacteria immediately before seeding it onto the plate or putting it into liquid worm cultures. Prepare the lipid-conditioned bacteria no longer than 1 h before feeding to the worms.
10. Mix the same volumes of filtered ethanol or DMSO (depending on which is used for the lipid feeding group) with BDR bacteria to serve as the vehicle control.

2. Preparation of synchronized  C. elegans for lipid supplementation

1. Prepare M9 buffer by mixing (using specified final concentrations) 22 mM KH₂PO₄, 22 mM Na₂HPO₄, 85 mM NaCl, and 2 mM MgSO₄ (see Table of Materials). Autoclave the buffer to sterilize.
2. Prepare a fresh bleach solution with 60% house bleach (v/v, see Table of Materials) and a final concentration of 1.6 M NaOH.
3. Collect gravid adults in a 15 mL conical tube using 10 mL of M9 buffer.
   NOTE: The number of worms and plates needed for synchronization depends on the experimental settings (i.e., number of conditions and replicates and feeding methods). Ideally, each full 6 cm plate of adult worms should be able to yield at least 600 L1 worms after synchronization.
4. Spin down the worms at 1,450 x g for 30 s. Remove the supernatant and rinse the worms one more time with 10 mL of M9 buffer.
5. Spin down the worms at 1,450 x g for 30 s. Aspirate the supernatant, leaving a 4 mL aliquot in the conical tube. Add 2 mL of bleach solution and shake vigorously for 1 min.
6. Spin down the worms at 1,450 x g for 30 s at room temperature.
7. Remove the supernatant and add 4 mL of M9 buffer and 2 mL of bleach solution. Shake the tube till the worm bodies are dissolved.
8. Spin down the eggs at 1,450 x g for 30 s at room temperature.
9. Remove the supernatant and wash the eggs with 10 mL of M9 buffer.
10. Repeat steps 2.8 and 2.9 3x.
11. Suspend the embryos with 6 mL of M9 buffer. Rock the embryos on a rotator at 20 °C for 2 days to let them hatch and synchronize.
12. Transfer L1 larvae to OP50 seeded plates. Incubate at 20 °C for 48 h to collect synchronized L4 worms, 72 h for day-1 adults, and 96 h for day-2 adults.
    NOTE: OP50 plates are prepared by adding 1 mL of 20x OP50 bacteria to each 10 cm nematode growth medium (NGM) plate³⁴. 30,000 L1 worms can be seeded to each of the OP50 plates if aiming to harvest the worms at the L4 stage, and 20,000 L1 worms can be seeded to each of the OP50 plates if aiming to harvest day-1 adults in the present experimental settings. It might be different for different lab settings, so it is recommended to test the worm numbers that can be seeded to ensure that the worms have enough food left before being harvested.
13. Collect L4, day-1 adult, or day-2 adult worms in a 15 mL conical tube using 10 mL of M9 buffer. Use another 5 mL of M9 buffer to rinse the leftover worms off the plates.
14. Spin down the worms at 1,450 x g for 30 s and discard the supernatant. Wash the worm pellet with 10 mL of BDR medium, centrifuge at 1,450 x g for 30 s, and discard the supernatant.
15. For worms in the liquid feeding method, transfer the BDR medium to the worms to achieve a worm concentration of 3,000 worms/mL. For worms in on-plate feeding methods, reduce the volume of BDR medium added to the worms to reduce the drying time on the plate.

3. Lipid supplementation for  C. elegans

1. Perform lipid supplementation in liquid culture following the steps below.
   NOTE: This method is suitable for testing transcriptional changes using bulk worms supplemented with lipids.
   1. Transfer the desired amount of lipid or vehicle control to each of the wells in a 12-well plate. Prepare three to four wells for each feeding condition as a biological replication.
   2. Mix the worm suspension from step 2.15 with 5x BDR bacteria in a 1:1 ratio to achieve a final concentration of 1,500 worms/mL and 2.5x for bacteria.
   3. Transfer 2 mL of the worm-bacteria mixture in BDR medium to each well of the 12-well plate.
   4. Wrap the 12-well plate with foil and shake in a 20 °C incubator at 100 rpm for the desired incubation length.
      NOTE: To test different feeding conditions, incubate the lipids with worms for different lengths of time, such as for 6 h, 12 h, and 24 h. In addition, different worm stages can be tested for optimal results, including lipid feeding from the L4 or day-1 adult stage.
2. Perform lipid supplementation on the 10 cm NGM agar plates following the steps below.
   NOTE: This method is suitable for testing transcriptional changes using bulk worms supplemented with lipids.
   1. Seed 1 mL of the lipid-conditioned bacteria from step 1.9 onto the center of each of the 10 cm plates. When a high number of plates have been prepared, vortex the final working solution multiple times in between seeding. Dry the plates in the dark using a biosafety hood.
   2. Transfer 3,000 worms from step 2.15 to each of the 10 cm plates. Dry the plates in a biosafety hood until the worms can crawl on the lipid-supplemented plates instead of swimming.
   3. Incubate the worms on the lipid-conditioned plates in a 20 °C incubator for the desired length of time. Protect from light if using poly-unsaturated lipids.
      NOTE: To test different feeding conditions, incubate the lipids with worms for various lengths of time, such as 6 h, 12 h, and 24 h. In addition, different worm stages can be tested for optimal results, including lipid feeding at the L4 or day-1 adult stage.
3. Perform lipid supplementation on the 6 cm NGM agar plates for transcriptional analysis.
   NOTE: This method is suitable for testing transcriptional changes in a few worms supplemented with lipids.
   1. Seed 300 µL of lipid-conditioned bacteria from step 1.9 onto the center of each of 6 cm plate. When a high number of plates have been prepared, vortex the final working solution multiple times in between seeding. Dry the plates in the dark using a biosafety hood.
   2. Transfer up to 300 worms from step 2.15 to each of the 6 cm plates. Dry the plates in a biosafety hood until worms can crawl on the lipid-supplemented plates instead of swimming.
   3. Incubate the worms on the lipid-conditioned plates in a 20 °C incubator for the desired length of time. Protect from light if using poly-unsaturated lipids.
      NOTE: To test different feeding conditions, incubate the lipids with worms at different time points, such as 6 h, 12 h, and 24 h. In addition, different worm stages can be tested for optimal results, including lipid feeding at the L4 or day-1 adult stage.
4. Perform lipid supplementation on the 6 cm NGM agar plates for longitudinal lifespan assay.
   1. Seed 200 µL of lipid-conditioned bacteria (with 1x lipid concentration and 5x concentrated bacteria) onto the center of each of the 6 cm NGM plates. Prepare three plates for each feeding condition.
      NOTE: The plates must be prepared freshly on the day of use (ideally right before use).
   2. Dry the plates in a biosafety hood. Keep lights in the hood and the room off if using poly-unsaturated lipids.
   3. Pick 30-40 synchronized L4 worms to each of the plates. Keep the plates with the worms in a 20 °C incubator. If feeding with poly-unsaturated lipids, keep plates in a light-protected box in the 20 °C incubator.
      NOTE: It is possible to use OP50 from a normal 6 cm passage plate (unconditioned OP50) as glue to pick up worms, but it is crucial to not leave a bulk amount of the unconditioned OP50 in the assay plate.
   4. Transfer the worms to new, freshly made lipid-conditioned plates daily or every other day, depending on the desired feeding condition. Survival is scored in the same way as previously described⁴.

4. RNA extraction for transcriptional analysis

1. Perform RNA extraction from a bulk number of whole worms.
   1. Transfer worms in the liquid culture from step 3.1 to 1.5 mL microcentrifuge tubes. Wash worms off the 10 cm plates from step 3.2 using M9 buffer and transfer the worms in M9 buffer to 1.5 mL microcentrifuge tubes.
   2. Briefly centrifuge the worms with a tabletop mini centrifuge (see Table of Materials) for 10 s and quickly aspirate the supernatant.
   3. Transfer leftover worms from the liquid feeding wells or wash from the plate feedings to the same tube and spin down. Remove the supernatant.
   4. Wash the worm pellets with 1 mL of ice-cold M9, briefly spin for 10 s with a mini centrifuge, and aspirate the supernatant. Repeat 1x or 2x depending on the transparency of the supernatant.
   5. Set the microcentrifuge tubes on ice for 2 min and remove as much supernatant as possible with a 200 µL pipette, leaving a packed worm pellet of no more than 15 µL volume.
   6. Transfer 15 µL of RNA extraction solution containing phenol and guanidine isothiocyanate (Table of Materials) to each of the microcentrifuge tubes and grind the worms with a motor grinder for about 30 s until no intact worms are visible.
      CAUTION: The RNA extraction solution is toxic and flammable, and any step involving an open container with this reagent must be operated in a chemical hood.
   7. Transfer 285 µL of the RNA extraction solution to the microcentrifuge tube while rinsing any worm content off the motor grinder tip to the microcentrifuge tube. Vortex to mix thoroughly.
      NOTE: This is a pause point. Samples can be stored in the RNA extraction solution at -80 °C for a couple of months. When taken out of the -80 °C, let samples thaw at room temperature.
   8. Transfer 60 µL of chloroform to each sample and vortex vigorously. Let the samples settle at room temperature for 10 min.
      CAUTION: Chloroform is toxic and must be handled in a chemical hood.
   9. Centrifuge at 21,000 x g for 20 min at 4 °C. Carefully transfer 140 µL of the aqueous layer on the top to a new and RNase-free microcentrifuge tube using a 200 µL pipette, transferring 2x with 70 µL each time.
      NOTE: From this step forward, all pipette tips and containers that have direct contact with the sample need to be RNase-free. It is also suggested to use RNase decontamination solution to wipe down all equipment and the working space.
   10. Transfer 140 μL of isopropanol to the sample tube. Vortex vigorously and let the sample settle at room temperature for 10 min. Centrifuge at 21,000 x g for 20 min at 4 °C, and carefully remove all supernatant.
   11. Transfer 0.5 mL of ice-cold 80% ethanol (v/v) to the sample tube with the RNA pellet. Vortex vigorously until the RNA pellet leaves the bottom of the tube and floats around in the ethanol solution. Centrifuge at 21,000 x g for 10 min at 4 °C and remove the supernatant.
   12. Briefly centrifuge the sample tubes in the mini centrifuge for 15 s to spin down any solvent adhering to the tube wall, and then remove the as much ethanol solution as possible with a pipette.
   13. Leave the tube cap open to dry the RNA pellet. If the pellet was washed thoroughly with the ethanol solution, the drying step should take less than 10 min.
       NOTE: This is a pause point. The dried RNA pellet can be stored at -80 °C for up to 1 month.
   14. Dissolve the dry RNA pellet with 40 µL of nuclease-free water and process with a DNA-removing kit according to the manufacturer instructions (see Table of Materials).
       NOTE: It is recommended to dissolve RNA in nuclease-free water first. If 40 µL of water is mixed with the 10x DNase buffer before transferring to the RNA tube, the RNA pellet will not dissolve completely. The RNA samples need to be kept on ice when dissolving the RNA pellet in water. Freeze-thaw cycles decrease the RNA quality; therefore, proceed to the reverse transcription step on the same day of the DNase treatment.
2. RNA extraction from a small number of whole worms.
   1. Pick 15-20 worms from their bacterial lawn into a fresh unseeded NGM plate to remove the bacteria from the worm.
   2. According to the manufacturer of the qRT-PCR 2-step kit (see Table of Materials), prepare 20 µL of final lysis solution for each sample by mixing 0.2 µL of DNase with 19.8 µL of lysis solution in PCR tubes. Mix the lysis solution by pipetting up and down 5x.
   3. Transfer 15-20 worms from the unseeded NGM plate into the PCR tube containing 20 µL of the final lysis solution with the minimum number of bacteria. Incubate the lysis reaction for 5 min at room temperature.
   4. Probe-sonicate (see Table of Materials) the worms with 30% amplitude using the following program: sonication for 5 s, pause for 5 s, and repeat 4x with a total sonication time of 20 s. Keep the samples in an ice-cold water bath during sonication.
   5. Incubate at room temperature for 5 min.
      NOTE: Add more DNase before incubation if the no-RT negative control shows DNA contamination at a concerning level.
   6. Transfer 2 µL of stop solution into the lysis reaction and mix by gently tapping. Incubate for 2 min at room temperature, and then set on ice.
      NOTE: The samples can be left on ice for up to 2 h before proceeding to the reverse transcription step.
3. Perform RNA extraction from worm tissues following the steps below.
   1. Pick around 20 worms from their bacterial lawn and place them onto a fresh unseeded NGM plate to remove the bacteria from them.
   2. Move the 20 worms (with as few bacteria as possible) to a watch glass containing 500 μL of M9 solution containing 4 µM levamisole (see Table of Materials). The immobilization will occur within seconds.
   3. When the worms are immobilized, dissect the germline or intestine using a 25 G needle that can be attached to the 1 mL syringe.
      1. Under the dissection scope, perform a cut at the position of the second pharyngeal bulb to allow natural extrusion of the intestine and germline. The two tissues can be easily distinguished based on their morphology and different contrast. Use tweezers or needles to gently separate the tissues, avoiding damaging them.
         NOTE: It is recommended to dissect within 5-10 min. If the cut does not produce a good tissue extrusion, it is suggested to move to the next worm and dissect as many as possible within 10 min. This procedure requires a short timeframe to avoid transcriptional changes from the environment.
   4. According to the qRT-PCR 2-step kit manufacturer, prepare 20 µL of final lysis solution for each sample by mixing 0.2 µL of DNase I to 19.8 µL of lysis solution in the PCR tubes. Mix the lysis solution by pipetting up and down 5x.
   5. Use an autoclaved glass pipette to transfer the dissected tissues into a PCR tube. Settle the PCR tubes on ice for 2 min to allow material deposition at the bottom. Remove the supernatant, add 20 µL of final lysis solution into the PCR tube, and mix by tapping.
   6. Incubate the lysis reaction for 5 min at room temperature. Then, transfer 2 µL of stop solution into the lysis reaction and mix by tapping. Incubate for 2 min at room temperature.
      ​NOTE: The samples can be left on ice for up to 2 h before proceeding to the reverse transcription step.

5. Reverse transcription and qRT-PCR

1. Perform reverse transcription and qRT-PCR from bulk worms.
   1. Measure the RNA concentration and prepare 5 µg of total RNA for reverse transcription.
   2. Prepare a pooled RNA sample by mixing equal amounts of RNA from each sample and adjusting the final RNA concentration to 0.556 g/L (5 µg/9 µL). Use the pooled RNA sample for the qRT-PCR standard curve and RT-negative controls.
   3. Perform reverse transcription according to manufacturer instructions (see Table of Materials). Run the following quality control reverse transcription reactions alongside the individual RNA samples.
      1. Perform reverse transcription with the pooled RNA sample as the template (for qRT-PCR standard curve).
      2. Perform reverse transcription with the pooled RNA sample as the template, without the reverse transcriptase (RT-negative control; quality control for potential genome DNA contamination).
      3. Perform reverse transcription with the nuclease-free water as the template (RT-negative control; quality control for potential RNA contamination in reagents).
         NOTE: This is a pause point. Samples can be left in the thermocycler overnight or stored at -20 °C for a few months.
   4. Dilute the cDNA generated from the pooled RNA samples four times, 20 times, 100 times, and 500 times with nuclease-free water for qRT-PCR standard curves.
   5. Dilute the cDNA generated from each RNA sample 20-100 times to use as templates for the qRT-PCR reactions.
      NOTE: The dilution ratio depends on the abundance of the gene of interest. For high-expressed genes, such as housekeeping genes or egl-3 and egl-21 used in this study, a 100x dilution is recommended. For low expressed genes, such as lbp-8, a 20x dilution is recommended.
   6. Perform qRT-PCR with qRT-PCR reagents according to the manufacturer instructions as follow: 95 °C for 10 min, repeat 40 times with 95 °C for 15 s and 60 °C for 1 min, followed by the default melting curve program, and hold at 8 °C for an indefinite time.
2. Perform reverse transcription and qRT-PCR from a few worms or worm tissues.
   1. Transfer 25 µL of RT buffer and 2.5 µL of 20x RT enzyme mix from the qPCR 2-step kit (see Table of Materials) into each tube containing worm lysate from either step 4.2.6 or step 4.3.6.
      NOTE: An RT-negative control is critical for qRT-PCR from a few worms. Each experiment needs to include a sample of worm lysate (from step 4.2.6) with RT buffer but without RT enzyme to examine the DNA contamination in the samples. If DNA contamination is an issue, consider adding more DNase to the lysis buffer or more DNase to the sample after the worms are lysed. Alternatively, one can perform RT-negative control for each sample by using half of the lysate in a normal RT reaction and the other half in an RT reaction without reverse transcriptase.
   2. Mix by gently tapping. Spin down using a mini centrifuge for 10 s.
   3. Load the samples into the thermocycler machine following the manufacturer's instructions: 37 °C for 60 min, 95 °C for 5 min, and 8 °C for an indefinite time.
      NOTE: This is a pause point. Samples can be left in the thermocycler overnight or stored at -20 °C for a few months.
   4. Keep all the reagents and samples on ice and protect the qRT-PCR reagents from light.
   5. Prepare a 96-well qPCR plate, and in each well mix 6 µL of nuclease-free water, 10 µL of qRT-PCR reagent, 2 µL of 5 µM primer mix (forward and reverse), and 2 µL of cDNA. Cover the 96-well qPCR plate surface with a protective optical film and press down to seal.
   6. Run the qRT-PCR program on a thermal cycler following the manufacturer's instructions as follows: 50 °C for 2 min, 95 °C for 2 min, repeat 40 times with 95 °C for 3 s and 60 °C for 30 s, followed by 8 °C for an indefinite time.

Representative Results

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 employed^7,8,33,35. The transcriptional induction of the neuropeptide processing genes egl-3 and egl-21 reported in previous studies^7,8,33 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-3^16,17. Previous studies have reported that fat-3 mutants lack 20-carbon PUFAs, including DGLA¹⁶. Previously, it was discovered that the loss of Δ6-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 intestines^36,37. 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 findings^36,38.

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.

Figure 1: Schematic of lipid feeding using different experimental settings. Please click here to view a larger version of this figure.

Figure 2: Validation of the neuropeptide processing genes' 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' egl-3 and egl-21 transcript levelsin lipl-4 Tg animals. Error bars represent ±1 SEM. **** p < 0.0001 by two-tailed Student's t-test. (B) RNA extraction from a few worms confirms the induction of neuropeptide processing genes' egl-3 and egl-21 transcript levels in lipl-4 Tg worms. Error bars represent ±1 SEM. **** p < 0.0001 by two-tailed Student's t-test. (C) Neuronal neuropeptide processing genes egl-3 and egl-21 are not induced in dissected intestines. Error bars represent ±1 SEM. Statistical analysis with two-tailed Student's t-test. (D) DGLA supplementation at different concentrations, including 10 µm, 100 µm, and 1 mM rescues the lipl-4Tg longevity effect upon fat-3 RNAi. *** p < 0.001 by log-rank test. This figure is adapted from Savini et al.³³. Please click here to view a larger version of this figure.

Discussion

Lipid supplementation has been employed in aging research to elucidate the direct impact of certain lipid species on healthy aging^{6,7,23,26,27,31}. 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 °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 µM 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 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.

Materials

1.5 mL Pestle	Genesee Scientific	93-165P15	For worm grinding with Trizol
Agarose	Sigma	A9639-500G
AmfiRivert cDNA Synthesis Platinum Master Mix	GenDEPOT	R5600	For reverse transcription from bulk worm samples
Applied Biosystems QuanStudio 3 Real-Time PCR	ThermoFisher	A28567	For qRT-PCR
Benchmark Scientific StripSpin 12 Microcentrifuge	Benchmark Scientific	C1248	For spin down PCR tubes
Branson 450 Digital Sonifier, w/ 1/8" tip	Branson Ultrasonic Corporation	100-132-888R
Chloroform	Fisher Scientific	C298-500
Cholesterol	Sigma	C8503-25G
Dimethyl sulfoxide (DMSO)	Sigma	D8418-100ML
Eppendorf 5424 R centrifuge	Eppendorf	22620444R	For RNA extraction
Eppendorf vapo protect mastercycler pro	Eppendorf	950030010	For reverse transcription
Ethanol, Absolute (200 Proof)	Fisher Scientific	BP2818-500
Greiner Bio-One CELLSTAR, 12 W Plate	Neta Scientific	665180	12-well plates for licuid feeding
Greiner Bio-One Petri Dish, Ps, 100 x 20 mm	Neta Scientific	664161	For bacterial LB plates and worm 10-cm NGM plates
Greiner Bio-One Petri Dish, Ps, 60 x 15 mm	Neta Scientific	628161	For worm6-cm NGM plates
Invitrogen nuclease-free water	ThermoFisher	AM9937
Isoproanol	Sigma	PX1835-2
Levamisole hydrochloride	VWR	SPCML1054
lipl-4Tg	MCW Lab	N/A	Transgenic C. elegans
lipl-4Tg;fat-3(wa22)	MCW Lab	N/A	Transgenic C. elegans
Luria Broth Base	ThermoFisher	12795-084
Magnesium sulfate (MgSO4)	Sigma	M2643-500G
MicroAmp EnduraPlate Optical 96-Well Fast Clear Reaction Plate with Barcode	ThermoFisher	4483354	96-well qPCR plate
MicroAmp Optical Adhesive Film	Applied BioSystem	4311971	For sealing the 96-well qPCR plate
Milli-Q Advantage A10 Water Purification System	Sigma	Z00Q0V0WW	Deionized water used to make all reagents, including buffer and cultural media, unless specified as nuclease-free water in the protocol
N2	Caenorhabditis Genetics Center	N/A	C. elegans wild isolate
NanoDrop ND-1000 Spectrophotometer	ThermoFisher	N/A	For measuring RNA concentration
OP50	Caenorhabditis Genetics Center	N/A	Bacteria used as C. elegans food
Potasium phosphate dibasic trihydrate (K2HPO4·3H2O)	Sigma	P5504-1KG
Potasium phosphate monobasic (KH2PO4)	Sigma	P0662-2.5KG
Power SYBR Green cells-to-Ct kit	ThermoFisher	4402953	For reverse transcription and qPCR from a few worms or worm tissue
Power SYBR Green Master Mix	ThermoFisher	4367659	For qPCR from bulk worm samples
Pure Bright germicidal ultra bleach	KIK International LLC.	59647210143	6% house bleach For worm egg preparation
Pyrex spot plate with nine depressions	Sigma	CLS722085-18EA	Watch glass for dissecting the worms
RNaseZap RNase Decontamination Solution	ThermoFisher	AM9780
Sodium cloride (NaCl)	Sigma	S7653-1KG
Sodium hydroxide (NaOH)	Sigma	SX0590-3
Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O)	Sigma	S9390-1KG
Thermo Sorvall Legend Mach 1.6R Centrifuge	Thermo	7500-4337	For bacteria collection
Thermo Sorvall ST 8 centrifuge	Thermo	7500-7200	For worm egg preparation
TRIzol Reagent	TheroFisher	15596018	RNA extraction reagent
Turbo DNA-free kit	ThermoFisher	AM1907	For removing DNA contamination in RNA extractions
Vortexer 59	Denville Scientific INV	S7030
VWR Disposable Pellet Mixers and Cordless Motor	VWR	47747-370	For worm grinding with Trizol
VWR Kinetic Energy 26 Joules Mini Centrifuge C1413 V-115	VWR	N/A	For worm collection. Discontinued model, a similar one available at VWR with Cat# 76269-064
Worm picker	WormStuff	59-AWP