The protocol provides a simple way to study American cockroaches. A common sanitary pest and a beneficial insect for traditional Chinese medicine. It will help to explore the molecular mechanisms of multiple life activities of American cockroaches by knocking down specific genes.
The main advantage of this technology is that it can knock down genes in American cockroach or other similar insects that are not suitable for gene editing. This technology can be used to explore various fields such as the genetic and epigenetic regulation, development process of tissues, regeneration of appendages, mating behavior, and other interesting sides of American cockroach. This technology is easy to operate and versatile.
We suggest to keep the animals healthy during DSRA injection treatment. I believe novices can easily master this technique after several practice sessions. To begin, separate the hatched nymphs from oothecae with a four millimeter aperture sieve.
Pick out the white freshly malted P.americana with a glass tube. Place the P.americana in new containers and wait for the correct stage for treatment. In the reaction system, add 10 microliters of T7 2X buffer, 2 microliters of the T7 Express Enzyme Mix and two micrograms of PCR product.
Finally, make up the total volume to 20 microliters with double distilled water. Gently mix upside down manually and incubate at 37 degrees Celsius for 30 minutes, then 70 degrees Celsius for 10 minutes. Dilute 4 micrograms per microliter RNase A solution with DEPC water at a ratio of 1 to 200.
Then add 1 microliter of one unit per microliter RQ1 RNase Free Dnase and 1 microliter of diluted RNase A solution to the system. Incubate at 37 degrees Celsius for 30 minutes. Then add 10%of the total volume of sodium acetate and three volumes of the total volume of isopropanol.
Gently mix upside down manually, then place on ice for five minutes and centrifuge at 13, 000 g for 10 minutes at 4 degrees Celsius. Remove the supernatant with a pipette. Next, wash the residue with 75%ethanol, with 25 percent DEPC treated water, then centrifuge at 13, 000 g for 5 minutes at 4 degrees Celsius.
Remove supernatant again, air dry the pellet for 15 minutes at room temperature. Then dissolve the double stranded RNA with about 100 microliters of DEPC water and dilute it to the final concentration of 2 micrograms per microliter. Set up the program in the microinjection pump to ensure that the volume of each injection is consistent.
Then clean the syringe by filling it with DEPC water 8 to 10 times. Install the 10 microliter syringe on the micro injection pump. Then start the pump and fill the syringe with 10 microliters of double stranded RNA solution.
Anesthetize the cockroaches with carbon dioxide, then gently pick them up with tweezers and deliver the cockroach toward the needle by hand. Next, insert the needle via the gap between two abdominal somites horizontally against the epidermis. Inject the double stranded RNA solution into the cockroach ensuring that the needle tip is as close as possible to the epidermis to avoid damaging internal organs.
Finally, pull out the needle tip. Put the injected cockroaches into clean bioassay containers. Wait for about 10 to 20 minutes to let them recover from the effects of carbon dioxide.
Label the containers with the date of injection, type and dose of double stranded RNA and age of the P.americana. Ddc or DOPA decarboxylase and Dpp or decapentaplegic genes were selected as two examples to observe the phenotype of the malted cockroaches after double stranded RNA injection and dsMocK which has no target in the animals was taken as a negative control. The RNAi efficiency was detected by qRT PCR.
The genes were significantly knocked down with a P value of less than 0.01 for dsDdc and less than 0.05 for dsDpp. P.americana with knockdown Ddc genes had the white epidermis due to blocked melanization. In the experiment with dsDpp injections, the RNAi disrupted limb regeneration.
The insects should be healthy and injected without harm. This technology provides a simple way to explore gene functions for insects that cannot be gene edited such as American cockroach.
