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Encyclopedia of Experiments

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


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.


This protocol is an excerpt from Groh et alMethods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegansJ. 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, 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 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.

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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
Image analysis software ImageJ
Analysis of mass spectrometry data Protein Prospector http://prospector.ucsf.edu/prospector/mshome.htm no
E.coli strains
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]


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