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. 2015 Jan 5:109:26.3.1-26.3.30.
doi: 10.1002/0471142727.mb2603s109.

RNA Interference in Caenorhabditis elegans

Darryl Conte Jr  1 , Lesley T MacNeil  2 , Albertha J M Walhout  2 , Craig C Mello  1   3
Affiliations

RNA Interference in Caenorhabditis elegans

Darryl Conte Jr et al. Curr Protoc Mol Biol. .

Abstract

RNAi has become an essential tool in C. elegans research. This unit describes procedures for RNAi in C. elegans by microinjecting with dsRNA, feeding with bacteria expressing dsRNA, and soaking in dsRNA solution, as well as high-throughput methods for RNAi-based screens.

Keywords: C. elegans; RNAi; feeding; high-throughput screening assays; microinjection; soaking.

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Figures

Figure 26.3.1
Figure 26.3.1
Loading the microinjection needle.
Figure 26.3.2
Figure 26.3.2
Attaching the microinjection needle to the microinjection assembly via tubing.
Figure 26.3.3
Figure 26.3.3
Overview of the microinjection technique. (A) The injection needle enters the field of view from the left of the image and is oriented above and roughly parallel to the body axis of the worm. (B to D) The needle is lowered until the tip touches the worm forming a slight indentation. The worm is pushed against the needle until the tip of the needle penetrates the cuticle. (E to G) The dsRNA solution is injected into the body cavity of the worm and flows in both directions away from the tip (indicated by the bars and arrows). (H) The needle is then backed out of the worm by moving the worm away from the needle.
Figure 26.3.4
Figure 26.3.4
Schematic of the zygotic RNAi procedure.
Figure 26.3.5
Figure 26.3.5
Multiple cloning site of the double T7 RNAi feeding vector L4440. Opposing phage T7 promoters flank the multiple cloning site of L4440. The restriction sites specific to the MCS are shown, and unique sites are indicated in bold.
Figure 26.3.6
Figure 26.3.6
Transferring a library from 96-well to 24-well plates. A 12-channel pipette loaded with tips on alternating channels is used to withdraw bacterial culture from the odd number wells in the top row of a 96-well deep-well plate (A) and transfer the cultures to the top row of a 24-well plate (B). Bacteria from the even number wells in the top row of the 96-well plate are then transferred to the second row of the 24-well plate. (C) Following this pattern, it takes two rows of a 96-well plate to fill a 24-well plate.
See this image and copyright information in PMC

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