RNAi Feeding in Liquid Culture: A High-Throughput Method to Knockdown Gene Expression in C. elegans
Published: April 30, 2023
Abstract
Quelle: Groh, N. et al. Methoden zur Untersuchung von Veränderungen in der inhärenten Proteinaggregation mit dem Alter in Caenorhabditis elegans. J. Vis. (2017).
RNAi ist ein leistungsstarkes genetisches Werkzeug, das C. elegans Forschern zur Verfügung steht. Dieses Video stellt eine Methode vor, um mehr Würmer mit RNAi zu behandeln als Plattenzuführen, indem flüssige Kulturbedingungen verwendet werden.
Protocol
Dieses Protokoll ist ein Auszug aus Groh et al, Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans, J. Vis. Exp. (2017). 1. Flüssige Kultur von gonadenlosen Tieren zur Sammlung von jungen und gealterten Tieren, die RNAi ausgesetzt sind und auf ein Gen von Interesse abzielen …
Materials
| Fernbach culture flask | Corning | 4425-2XL | Pyrex, Capacity 2,800 ml, with 3 baffle indents | yes |
| Membrane Screw Cap | Schott | 1E+06 | GL45 | yes |
| Nutating Mixer | VWR | 444-0148 | yes | |
| Separatory funnel | Nalgene | 4300-1000 | Capacity 1,000 ml | no |
| 1 ml syringe | BD Plastipak | 3E+05 | no | |
| Gray needle, 27 G x ½ ", 0.4 mm x 13 mm | BD Microlance 3 | 3E+05 | no | |
| Membrane filters 0.025 µM | Millipore | VSWP04700 | no | |
| pH strip | Machery-Nagel | 92110 | pH-Fix 0-14 | no |
| Protease Inhibitor Cocktail | Roche | 5E+09 | Complete Mini EDTA-free tablets | no |
| Octoxynol-9 | Applichem | A1388 | Triton X-100 | no |
| 4-Morpholineethanesulfonic acid (MES) | Sigma-Aldrich | M1317 | no | |
| Nonylphenylpolyethylenglycol | Applichem | A1694 | Nonidet P40 (NP40) | no |
| DNaseI | Roche | 5E+09 | recombinant, RNase free | no |
| RNaseA | Promega | A7973 | solution | no |
| Total protein blot staining | Thermofisher | S11791 | Sypro Ruby protein blot stain | no |
| Total protein gel staining | Thermofisher | S12001 | Sypro Ruby protein gel stain | no |
| TCEP (tris (2-carboxyethyl) phosphine hydrochloride) | Serva | 36970 | no | |
| Iodoacetamide | Serva | 26710 | no | |
| Ammoniumbicarbonate | Sigma-Aldrich | 9830 | no | |
| Sequencing Grade Modified Trypsin | Promega | V5111 | no | |
| Isobaric tags for relative and absolute quantitation | Sciex | 4E+06 | iTRAQ Reagents Multiplex Kit | no |
| Centrifuge Avanti J-26XP | Beckmann Coulter | 4E+05 | yes | |
| Ultracentrifuge Optima Max-XP | Beckmann Coulter | 4E+05 | no | |
| Centrifuge 5424R | Eppendorf | 5E+09 | yes | |
| Centrifuge 5702 | Eppendorf | 6E+09 | yes | |
| Centrifuge Megafuge 40R | Thermo Scientific | 8E+07 | no | |
| Concentrator Plus | Eppendorf | 5E+09 | Centrifugal evaporator | no |
| Fluorescent stereo-microscope M165 FC | Leica | With Planapo 2.0x objective | no | |
| Dissection microscope | Leica | Leica S6E | yes | |
| High magnification microscope Zeiss Axio Observer Z1 | Zeiss | With PlanAPOCHROMAT 20x objective and Zeiss Axio Cam MRm | no | |
| Software | no | |||
| Image analysis software | ImageJ | no | ||
| 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] | ||
Cite This Article
RNAi Feeding in Liquid Culture: A High-Throughput Method to Knockdown Gene Expression in C. elegans. J. Vis. Exp. (Pending Publication), e20139, doi: (2023).