to induce dsRNA synthesis in the ..

T1 - RNA interference by feeding in vitro-synthesized double-stranded RNA to planarians
Photo provided by Flickr

for in vitro dsRNA synthesis and transfection into Sf cells ..

As for the WISH protocol, the first steps of the RNAi protocol in planarian are: look for mRNA species-specific sequences that correspond to the genes of interest and design F and R primers () as described in , produce the cDNA and clone the gene of interest as described in , transform the bacteria and sequence the insert as described in .

For example, chemically synthesized or in vitro transcribed siRNAs can be transfected into cells, injected into mice, or introduced into plants.
Photo provided by Flickr

Protocol for Double-Stranded RNA Synthesis.

Methods for expressing siRNAs in cells in culture and in vivo using viral vectors, and for transfecting cells with synthetic siRNAs, have been developed and are being used to establish the functions of specific proteins in various cell types and organisms.

cyclic RNA; RNA synthesis; short interfering RNA; RNA interference; dumbbell‐shaped structure; ligase
Photo provided by Flickr

Pol η has the ability to catalyze translesion synthesis (TLS) past UV-induced cyclobutane pyrimidine dimers (CPDs) and some otherlesions as well. Loss of pol η in humans results in increased photosensitivity and the cancer-prone genetic disorder xeroderma pigmentosum variant (XPV). We provide an example of the feeding RNAi technique, in which downregulation of pol η in C. elegans results in increased sensitivity of several development anddifferentiation processes, including meiosis and embryogenesis to UVradiation.

Following second strand synthesis, transfer contents of 0.5 ml microfuge into a 1.5 ml RNase-free microfuge tube.
Photo provided by Flickr


Role of RNA in Protein Synthesis ..

N2 - Background: The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced by means of injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA. Results: We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery. Conclusions: This method gives robust and reproducible results and is amenable to high-throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems. Developmental Dynamics 242:718-730, 2013.

"RNA Interference by Feeding In Vitro–Synthesized …

RNAi is widely used by researchers to silence genes in order to learn something about their function. siRNAs can be designed to match any gene, can be manufactured cheaply, and can be readily administered to cells. One can now order commercially synthesized siRNAs to silence virtually any gene in a human or other organism's cell, dramatically accelerating the pace of biomedical research. Furthermore, the ability to turn off expression of a single gene makes RNAi an appealing therapeutic approach to treat infectious diseases or genetic disorders, such as those that result from the inappropriate and undesirable activity of a gene, as in many cancers and neurodegenerative diseases. There are currently several clinical trials testing the safety and effectiveness of siRNA drugs.

Role of RNA in Protein Synthesis

Double-stranded RNA (dsRNA)-induced gene silencing in Caenorhabditis elegans involves the manufacture and delivery of defined sequences of dsRNA to the organism, followed by a careful monitoring for loss-of-function phenocopies in treated animals. In this chapter, we describe how to generate DNAs that can be used as templates for transcription of dsRNA.