Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)
Summary
Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.
Abstract
Protocol
Discussion
Efficiency of gene silencing is highly variable and depends on a number of factors including transcript and protein turn-over rates, and dsRNA uptake efficiency by cells and organs. We have found that silencing begins as early as 1 day post injection and can last up to 6 days post injection. Use transcript and protein detection methods such as quantitative PCR and Western blotting to validate silencing.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Megascript | kit | Ambion | for dsRNA synthesis; T7 RNA polymerase-mediated in vitro transcription reaction | |
| RNeasy | Qiagen | For dsRNA purification | ||
| dsRNA | should be quantified and adjusted to 3 μg/ μL in water | |||
| fine-tipped forceps | ||||
| glass slide | ||||
| glass microcapillary needles | ||||
| microinjector | Drummond | Nanoject II | ||
| light microscope | mounted above a cold block | |||
| Petri dish | covered, glass | |||
| paper towels | ||||
| 10% sucrose | food | |||
| A. gambiae | Animal | mosquitos |
