Applications of RNA Interference in American Cockroach

Summary

The present protocol describes step-by-step guidelines for the RNAi operation techniques in P. americana.

Abstract

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous characteristics. Altogether with well-annotated genome sequences, these advantages have made American cockroach, Periplaneta americana, an important hemimetabolous insect model. Limited by the shortage of knockout strategy, effective RNA interference (RNAi)-based gene knockdown becomes an indispensable technique in functional gene research of P. americana. The present protocol describes the RNAi operation techniques in P. americana. The protocol includes (1) selection of the P. americana at proper developmental stages, (2) preparation for the injection setting, (3) dsRNA injection, and (4) gene knockdown efficiency detection. RNAi is a powerful reverse genetic tool in P. americana. The majority of P. americana tissues are sensitive to extracellular dsRNA. Its simplicity allows researchers to quickly obtain dysfunctional phenotypes under one or multiple targeting dsRNA injections, enabling researchers to better use the P. americana for developmental and metamorphic studies.

Introduction

RNA interference (RNAi), an evolutionarily conserved mechanism, gradually becomes an essential reverse-genetic tool to inhibit gene expression in many organisms¹, since Andrew Fire and Craig Mello² developed the double-stranded RNA (dsRNA) mediated gene silence strategy. dsRNA is cleaved into fragments of 21-23 nucleotides, small interfering RNAs (siRNAs), by the enzyme Dicer in cells to activate the RNAi pathway. Then siRNAs are incorporated into the RNA-induced silencing complex (RISC), which couples to the target mRNA, causes mRNA cleavage, and finally results in the loss of gene function^3....

Protocol

The line of P. americana was initially provided by Dr. Huiling Hao. This species has been maintained with inbreeding for 30 years⁹.

1. Hatching and feeding of P. americana

1. Collect fresh oothecae (immediately post egg-laying) of P. americana and incubate in the dark incubator at 25 °C and 60% humidity for ~25 days. Then increase the temperature and humidity to 30 °C and 75% 3 days before hatching.
2. Use a sieve with 4 mm aperture to separate the hatched nymphs from oothecae.
3. Keep the nymphs in cylindrical containers (12 cm in diam....

Results

Figure 1 shows a successful injection. The microinjection syringe with a micro diameter needle should be horizontally placed on the booster (Figure 1A). The needle is inserted via the gap between two abdominal somites horizontally against the epidermis (Figure 1B). Ensure that the liquid goes into the P. americana abdomen. The too steep angle of the needle will damage the internal organs (Figur.......

Discussion

This report described a methodological step-by-step RNAi strategy in P. americana; of note, it also can be applied to other cockroaches (Blattella germanica, for example) and many other insects with minor changes. However, the gene silencing efficiency of RNAi is not always high enough, with an obvious disadvantage compared with the gene knockout strategy¹³. The following residual effect of gene-level may interfere with the real phenotypes. To ensure the RNAi treatment is success.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
701 N 10 µL Syr (26s/51/2)	Hamilton	PN:80300	Injection
Incubator	Ningbo Jiangnan Instrument Factory	RXZ-380A-LED	For cockroaches hatching and feeding
Micro-injection pump	Alcott Biotechnology	ALC-IP600	Injection
pTOPO-Blunt Cloning Kit	Aidlab Biotechnology	CV16	For Gene clonging
quantitative Real-Time PCR Systems	Bio-Rad	CFX Connect	For qRT-PCR analysis
T7 RiboMAX Express RNAi System	Promega	P1700	For dsRNA synthesis, which contains Rnase A Solution (4 μg/μL), Sodium Acetate, 3.0M (pH 5.2), Enzyme Mix, T7 Express, Nuclease-Free water, Express T7 2x Buffer, RQ1 RNase-Free DNase
Thermal Cyclers	Bio-Rad	S1000	For DNA amplification