I discussed the theory of RNA interference here.
In short, RNA interference induces the knock down of a gene by injecting double stranded RNA. Here I will show how we inject this double stranded RNA in Tribolium. We inject the RNA into pupa, this while they are easy to handle. However, it is also possible to inject RNA into the eggs, larva or adults.




First we have to gather the pupa by sieving them out of the flour. We are looking for pupa that are almost finished developing and are ready to hatch as adults. We can recognize them by their darkened wings and dark/red mouthparts (red arrows). After we collected pupa of the right stage we determine their sex. We do this because only females produce eggs and we are trying to discover the function of a gene in the egg.

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Because we only want to inject females, we are going to determine the sex of the pupa. We can do this by looking at their genitalia, females have paired genitalia that are clearly visible while males do not have these (see figure below, genitalia are highlighted in orange).

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After sexing the pupa we are ready to glue them to a microscope slide.

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Next, we load the glass needle with double stranded RNA (colored red by Vital Red dye):

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Then we inject the pupa under the microscope:

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When were finished injecting them, we put the slides upside down in flour. As soon as they hatch the beetles will fall into the flour. The other pupa in front of the glass slides are the males, these are added without injecting them to fertilize the females.

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In both the females we injected as in the eggs they will lay, the gene has been knocked down. Now we can figure out what the function of this gene is. Here is a short movie of RNA interference in Tribolium.

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