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.

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 th.......

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.......

Materials

Name	Company	Catalog Number	Comments
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