Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The overall goal of this procedure is to knock down a gene of interest via RNA interference in the red flower beetle trium cadium by injecting double stranded RNA solution into the larval body cavity. This is accomplished by first isolating the proper stage of the larvae from the culture by sifting the culture flower and separating the larvae from other stages of beetles on a seed pan. The second step is to prepare an injection needle by breaking the tip of the needle, placing it onto a needle holder and front loading, double stranded RNA solution of desired concentration into the needle.

Next, authorize the isolated larvae and place the larvae onto a sticky glass slide. The final step is to inject the larvae with a double stranded RNA solution under a dissecting microscope. Ultimately, RNAi related phenotypes at the desired stage are analyzed to determine the function of the targeted gene.

The main advantage of this technique over existing method, such as neurogenesis or transgenesis, is that just a simple injection of doublet stranded, RNA solution into the beetles body cavity can induce efficient and long lasting gene knockdown at any desired stage of bedo development. Although the protocol described here will focus on trium castin larvae with some modification, the injection procedures can be applied to other stages of trium, castin, and other insects. Generally, individuals new to this method will struggle with making a good needle and injecting the appropriate amount of double strand RNA solution without killing the larvae.

Visual demonstration of this technique is critical as the doublet stranded RNA injection and needle preparation steps are difficult to describe and can only be learned through observation and practice Culture. The tea cast strain of choice as detailed in the accompanying text protocol. Empty one culture bottle of the beetles onto a number 25 sieve immediately and aggressively sift to collect the older larvae, pupi, and adults.

Then transfer the beetles along with exus to a seed pan tapping to prevent the beetles from escaping. Now, gently blow over the seed pan to remove the exus and the flower particles from the surface of the beetles. Tap the seed pan to settle.

The contents. Wait for the more mobile adults to come out from the pile and remove them to separate the pupi angle, the pan slightly downward. Gently knock to roll and remove the pupi, leaving only the larvae on the seed pan.

Now place the isolated larvae in a clean Petri dish. Select the desired larval stage for injection under a microscope, and keep them in a Petri dish until authorization. Combine the remaining stages of the beetles and place them back in a culture bottle.

Attach a pulled glass needle onto a glass microscope. Slide under a dissecting microscope. Locate where the tip bends.

Grab the area slightly toward the tip side and twist to break for optimal penetration of the larval cuticle, select a needle with an angled opening of approximately 0.05 millimeters in diameter. Now unscrew the tip of the needle holder. Place the back end of the needle into the holder tip and the rubber gasket under the holder tip.

Then insert the back of the needle into the holder, ensuring that the rubber gasket is inserted into the opening. Screw the tip back onto the holder. Now place the assembled needle holder onto the needle manipulator.

Adjust the position so that the tip of the needle is centered under the microscope field of view. Prepare the double stranded RNA solution just prior to injection. Cut the tip of a 20 microliter disposable pipette tip.

Now pipette 10 microliters of the solution into the trimmed pipette tip. Carefully remove the pipette tip from the pipette while keeping the fluid at the tip by providing back pressure. Place the pipette tip with the double stranded RNA solution on the microscope stage.

Looking through the microscope slowly move the loaded tip toward the needle until the tip of the needle is just inside the loaded pipette tip and into the solution. Then gently pull back on the plunger of the injection syringe to slowly pull the solution into the needle without overloading the needle. As the end of the DS RNA solution approaches the tip of the needle, slow the rate of pulling and stop loading the solution Before the tip of the needle would reach air to avoid rapid uptake into the needle holder.

Open the stop cock to remove the pressure from the injection syringe and needle holder. Then move the pipette tip away from the tip of the needle. Now raise the needle up the stage and remove the emptied pipette tip from the microscope stage.

Place the larvae into the e authorization basket and then place the basket in the ether bottle and close the lid. After three minutes, check the larvae for movement. If they are still moving, place them back in the ether bottle for another 30 seconds.

Transfer the E authorized larvae from the basket to the non-stick edge of a sticky glass slide. Transfer the larvae sequentially to the slide. Lay each one laterally.

Gently tap down the body from head to tail with forceps and slightly stretch it to secure the larvae on the slide. Now position the prepared slide on the microscope stage. Insert the double stranded RNA loaded needle gently into the dorsal side of the larvae, using the stage to move the larvae to the needle.

Then pull the needle slightly back for the DS RNA. To enter the larva body cavity, push gently on the injection syringe until the larvae turned green and look stretched and full. Next, remove the needle from the larvae while slightly pulling back on the injection syringe.

Inject all larvae on the slide. Make sure to remove larvae that were not successfully injected. Set the injected larvae aside until all larvae recover from the E authorization.

Now gently lift the larvae from head to tail transfer to a clean Petri dish and leave them at least 10 minutes to allow the injection wound to clot. Then culture, the injected larvae at 30 degrees Celsius with 70%humidity. Observe the larvae for RNAI phenotypes regularly.

These examples show varying success levels of injections with green double stranded RNA solution into the last instar larva with a properly sized injection needle. The needle tip penetrates the larva cuticle with minimal resistance, and the green DS RNA solution flows into the larvae without any leakage. In the PU 11 strain, the enhanced yellow fluorescent protein can servee as a good positive control for double stranded RNA injection experiments.

When EYFP DS RNA is injected in the last larval stage, a significant reduction in EYFP signal in the future wing primordial can be observed as early as one day post-injection. The knockdown of EYFP expression is generally observable two days after injection, and if the DS RNA concentration is high enough will persist through the pupil stage and throughout the life of the beetle. Since EYFP is a non endogenous gene sequence, this gene can also be used as a negative control that should not cause morphological or physiological disruptions.

In this experiment, the double stranded RNA for vestigial, a critical wing gene was injected into penultimate stage larvae. Note that complete loss of the wing structures can be observed in the pupil stage. The resulting adult also completely lacks wing structures.

Once master, this technique can be done in less than two hours if it is performed properly Following this procedure. Other methods such as quantitative real-time PCR in situ hybridization or immunohistochemistry can be performed in order to answer additional questions like what genes are regulated by the targeted gene After its development. This technique paved the way for researchers to perform loss of function analysis and post embryonic stages in traum casten without utilizing complex genetics techniques.

The simple nature of this method also makes it highly applicable to a teaching setting. After watching this video, you should have a good understanding of how to select the appropriate stage of love for injection, how to make an injection needle, and perform allowable injection of double standard RNA successfully. This protocol will allow you to evaluate the function of Gene through level RNI in Trium.