RNAi Feeding in Liquid Culture: A High-Throughput Method to Knockdown Gene Expression in C. elegans

Abstract

Source: Groh, N. et al. Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans. J. Vis. Exp. (2017).

RNAi is powerful genetic tool available to C. elegans researchers. This video introduces a method to treat more worms with RNAi than plate feeding by utilizing liquid culture conditions.

Protocol

This protocol is an excerpt from Groh et al, Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans, J. Vis. Exp. (2017).

1. Liquid culture of gonad-less animals to collect young and aged animals subjected to RNAi targeting a gene of interest

NOTE: The response of some genes to RNAi can be temperature dependent as previously described. As an alternative to RNAi, a mutant gene could be incorporated into the gon-2(-) background.

1. Preparation of bacteria for liquid culture
   1. Prepare OP50 and RNAi bacteria (bacteria producing the desired dsRNA and bacteria with the empty vector as control) by inoculating 4 L LB medium with 12 mL of the respective bacterial culture (add a final concentration of 50 µg/mL carbenicillin and 1 mM IPTG to the RNAi bacterial cultures) and incubate them at 37 °C overnight at 180 rpm.
   2. The next day harvest the cultures at 6,700 x g for 10 min at 4 °C. Remove the supernatants and resuspend each pellet in 60 mL ice-cold S basal media (100 mM NaCl, 50 mM Potassium phosphate pH 6, kept on ice). Keep the three resuspended pellets (OP50, control RNAi bacteria, and RNAi bacteria for the gene of interest) at 4 °C until step 1.2.
      NOTE: Worms can be grown on RNAi from L1 stage or to avoid developmental defects from the last larval stage L4 (see step 1.2).
2. Liquid culture for treatment with RNAi starting at last larval stage L4
   1. Add 200 mL S basal media in a Fernbach culture flask (capacity 2,800 mL). For a final culture volume of 300 mL (see 1.2.2.4), add 10 mM potassium citrate, pH 6, 3 mL Trace metals solution (5 mM ethylenediaminetetraacetic acid (EDTA), 2.5 mM FeSO₄, 1 mM MnCl₂, 1 mM ZnSO₄, 0.1 mM CuSO₄), 3 mM MgSO₄, 3 mM CaCl₂, 100 ng/mL carbendazim, and 5 µg/mL cholesterol). Close the flask with a membrane screw cap. Refer to Table 1 for recipes of buffers.
   2. Take the L1s out of 25 °C and transfer them to 15 mL tubes. Centrifuge at 1,900 x g for 3 min, remove the supernatant, and count the L1s per 2 µL. Add 500,000 L1s into the Fernbach culture flask prepared in the previous step.
   3. Add OP50 (from step 1.1) proportionally to the number of worms. For example, for 500,000 worms add 60 mL OP50.
   4. Complete worm culture with S basal to bring the total volume to 300 mL. Incubate the worm culture until L4 stage at 25 °C in a shaking incubator with 150 rpm.
   5. The next day, the worms should be the size of wild-type L4s. To change from OP50 to RNAi bacteria, collect animals in six 50 mL-tubes, let them sediment and remove the supernatant. Wash the L4s with M9 to remove residual OP50 bacteria: centrifuge at 1900 x g for 3 min, remove the supernatant and transfer all L4s into one 50 mL-tube. Count the L4s per 5 µL (at least nine times). Two times 50,000 L4s are needed for the young worm collection and two times 100,000 L4s are needed for the aged worm collection.
   6. Prepare four Fernbach culture flasks as described in 1.2.2.1. Add also a final concentration of 50 µg/mL Carbenicillin and 1 mM IPTG to each flask.
   7. Add control RNAi bacteria and RNAi bacteria for the gene of interest (from step 1.1) proportionally to the number of worms: Add 7 mL of the respective bacteria per flask for young worms and 14 mL per flask for the aged worms.
   8. Add 50,000 worms per flask for the young worm collection and 100,000 worms per flask for the aged worm collection, and complete the cultures with S basal to fill up to 300 mL. Incubate the worm cultures (four in total) at 25 °C and 150 rpm.

2. Maintenance of C. elegans liquid cultures

1. Periodically collect an aliquot from each liquid culture and place on a glass slide (alternatively on an agar plate) to verify that there are no contaminations. Using a dissection microscope, check the bacterial food levels in the culture especially during the growth phase. Prepare in advance 2 L cultures of control RNAi bacteria and RNAi bacteria for the gene of interest as described in step 1.1. Add these to C. elegans liquid cultures if necessary and keep the rest at 4 °C.
2. Periodically check that the animals are sterile. The majority of animals will not have a gonad. Depending on the penetrance of the gon-2 mutation, some may have aborted gonadal structures but no animals with eggs should be observed.

Stock solution	Amount	Final concentration	Comments/Description
S basal (For 500 mL: 5.9 g NaCl, 50 mL 1 M Potassium phosphate pH 6)	200 mL		1 M Potassium phosphate pH 6: For 1 L: 129 g KH2PO4 monobasic, 52 g K2HPO4 dibasic anhydrous
1 M Potassium citrate pH 6 (For 400 mL: 13.1 g citric acid monohydrate, 134.4 g tri-potassium citrate monohydrate)	3 mL	10 mM
Trace metals solution (For 1 L: 1.86 g Ethylenediaminetetraacetic acid (EDTA), 0.69 g FeSO4 · 7 H2O, 0.2 g MnCl2 · 4 H2O, 0.29 g ZnSO4 · 7 H2O, 0.025 g CuSO4 · 5 H2O)	3 mL		Store stock solution in the dark at 4 °C
1 M MgSO4 (For 500 mL: 123.3 g)	900 µL	3 mM
1 M CaCl2 (For 500 mL: 73.5 g)	900 µL	3 mM
200 µg/mL Carbendazim (For 50 mL: 10 mg in Methanol)	150 µL	100 ng/mL
5 mg/mL Cholesterol (For 100 mL: 0.5 g in Ethanol)	300 µL	5 µg/mL

Table 1.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fernbach culture flask	Corning	4425-2XL	Pyrex, Capacity 2,800 ml, with 3 baffle indents
Membrane Screw Cap	Schott	1E+06	GL45
Nutating Mixer	VWR	444-0148	yes
Separatory funnel	Nalgene	4300-1000	Capacity 1,000 ml
1 ml syringe	BD Plastipak	3E+05
Gray needle, 27 G x ½ ", 0.4 mm x 13 mm	BD Microlance 3	3E+05
Membrane filters 0.025 µM	Millipore	VSWP04700	no
pH strip	Machery-Nagel	92110	pH-Fix 0-14
Protease Inhibitor Cocktail	Roche	5E+09	Complete Mini EDTA-free tablets
Octoxynol-9	Applichem	A1388	Triton X-100
4-Morpholineethanesulfonic acid (MES)	Sigma-Aldrich	M1317
Nonylphenylpolyethylenglycol	Applichem	A1694	Nonidet P40 (NP40)
DNaseI	Roche	5E+09	recombinant, RNase free
RNaseA	Promega	A7973	solution
Total protein blot staining	Thermofisher	S11791	Sypro Ruby protein blot stain
Total protein gel staining	Thermofisher	S12001	Sypro Ruby protein gel stain
TCEP (tris (2-carboxyethyl) phosphine hydrochloride)	Serva	36970
Iodoacetamide	Serva	26710
Ammoniumbicarbonate	Sigma-Aldrich	9830
Sequencing Grade Modified Trypsin	Promega	V5111
Isobaric tags for relative and absolute quantitation	Sciex	4E+06	iTRAQ Reagents Multiplex Kit
Centrifuge Avanti J-26XP	Beckmann Coulter	4E+05
Ultracentrifuge Optima Max-XP	Beckmann Coulter	4E+05
Centrifuge 5424R	Eppendorf	5E+09
Centrifuge 5702	Eppendorf	6E+09
Centrifuge Megafuge 40R	Thermo Scientific	8E+07
Concentrator Plus	Eppendorf	5E+09	Centrifugal evaporator
Fluorescent stereo-microscope M165 FC	Leica		With Planapo 2.0x objective
Dissection microscope	Leica		Leica S6E
High magnification microscope Zeiss Axio Observer Z1	Zeiss		With PlanAPOCHROMAT 20x objective and Zeiss Axio Cam MRm
Software
Image analysis software	ImageJ
Analysis of mass spectrometry data	Protein Prospector	http://prospector.ucsf.edu/prospector/mshome.htm	no
E.coli strains
OP50	CGC
RNAi bacteria
L4440	Julie Ahringer RNAi library
C. elegans mutants
CF2253	CGC, strain name: EJ1158	Genotype: gon-2(q388)
C. elegans transgenics
DCD214	Della David's lab at DZNE Tübingen	Genotype: N2; uqIs24[Pmyo-2::tagrfp::pab-1]
DCD215	Della David's lab at DZNE Tübingen	Genotype: daf-2(e1370) III; uqIs24[Pmyo-2::tagrfp::pab-1]