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.
