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Tools & Techniques: Enhanced dsRNA Uptake in Insects, microRNA Detection at the Single-Cell Level, and More


Researchers from the Chinese Academy of Sciences have reported on a new discovery that could help enhance plant-mediated RNAi defense strategies against herbivorous insects.

While oral ingestion of plant-expressed dsRNA can trigger target gene suppression in insects, the insect peritrophic matrix, which prevents large molecules from passing into midgut cells, is a key barrier to the process, according to the investigators.

The team found that uptake of plant cysteine proteases, such as GhCP1 from cotton and AtCP2 from Arabidopsis, by cotton bollworm larvae attenuated the peritrophic matrix. In a paper appearing in Plant Molecular Biology, they also showed that larvae pre-fed with either of the cysteine proteases and transferred to a gossypol-containing diet accumulated higher content of gossypol in their midguts.

Additionally, larvae that had previously ingested GhCP1 or AtCP2 were more susceptible to infection by the dsRNA virus Dendrolimus punctatus cytoplasmic polyhedrosis virus. “Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant,” the researchers wrote.

By crippling the insect peritrophic matrix, plant cysteine proteases may prove to be a key tool in optimizing RNAi-based approaches for plant protection, the team concluded. “Furthermore, the cysteine proteases may also play a role in affecting various proteins in midgut, disturbing the insect digestion and even defense systems” — an area of research that requires additional investigation.

Aiming to overcome limitations of using Caenorhabditis elegans for studying the mechanisms of aging, a research team from the Chinese Academy of Sciences has created an automated, integrated microfluidic device for culturing nematodes and demonstrated its utility in RNAi experiments.

Dubbed WormFarm, the approach involves maintaining the animals throughout their life spans in separate containers on a single polydimethylsiloxane chip, which includes design features that enable automated removal of progeny and efficient control of environmental conditions, according to a report in Aging Cell.

“In addition, we have developed computational algorithms for automated analysis of video footage to quantify survival and other phenotypes, such as body size and motility,” the investigators wrote.

As a proof of concept, the team used WormFarm to recapitulate survival data obtained from a standard plate-based assay for RNAi-induced changes in C. elegans life span.

According to the paper, the animals were aged in WormFarm and then fed bacteria containing either an empty-vector control plasmid or RNAi plasmids against three targets, and found that life spans obtained from the nematodes “showed the expected trends and matched the survival” of animals aged on nematode growth media plates.

“WormFarm provides many advantages over traditional methods, including alleviating contamination by progeny without use of drugs that inhibit reproduction; providing improved environmental control; removing the potential for unintentional human bias by automated acquisition and analysis of images and movies; and dramatically reducing costs associated with consumption of reagents and chemicals,” the scientists concluded.

A team from Ohio State University has published details of a new method to ready nucleic acids for delivery using lipoplexes prepared in microwell arrays.

Specifically, the researchers distributed nucleic acid-containing lipoplexes uniformly in a polydimethlysiloxane microwell array using a discontinuous de-wetting technique and without any surface modifications to the polydimethlysiloxane or chemical/biological modifications to the lipoplexes, they wrote in Small.

The resulting lipoplexes were disc-like in shape and had a diameter of around 817.6 nanometers and a thickness of around 194.5 nanometers, and microwell array-mediated delivery of the lipoplexes to an adherent cell line and a suspension cell line was significantly more efficient than conventional transfection methods, according to the paper.

To demonstrate the therapeutic efficacy of the lipoplexes delivered via the microwell array, the investigators used microRNA-29b, which is down-regulated in lung tumors, as a model nucleic acid, the paper states. They found that the effective therapeutic dosage of the miRNA in vitro was reduced from the microgram level with traditional transfection to the nanogram level with the lipoplexes.

“In the future, the microwell array may be combined with various transfection reagents to improve in vitro transfection efficiency and achieve spatial and temporal controlled delivery, which may have great potential in applications such as controlled stem cell differentiation,” the researchers wrote. “In vivo, the microwell array delivery system may be incorporated with transplants, stents, and scaffolds to deliver therapeutic reagents for tissue engineering, or be used in transdermal delivery of vaccine and therapeutics.”

Investigators from Huazhong University have published the details of a new ultra-sensitive method for microRNA detection at the single-cell level from cancer cell lines and clinical samples.

While Northern blotting and microarrays are commonly used for miRNA quantification, improving the sensitivity and specificity of these approaches is hindered by the small size and low abundance of the non-coding RNAs, as well as sequence homology among family members, the scientists wrote in The Journal of the American Chemical Society. Meantime, amplification strategies such as real-time PCR suffer from their own shortcomings.

To address this issue, the researchers developed a one-pot hairpin-mediated quadratic enzymatic amplification strategy for miRNA detection, which is not only designed to sidestep the limitations of other methods, but allow direct miRNA detection in crude cellular extracts of cancer cells and tissues.

The technique is based on polymerase-aided strand-displacement polymerization and exonuclease-assisted template recycling, which “achieves rapid, isothermal, and highly sensitive detection of miRNAs,” according to the paper.

Additionally, the assay “requires only one step to realize quadratic amplification for ultra-high sensitive detection of miRNAs, [and] there is no need for multiple self-assembly steps required in nanoparticle-based amplification [assays] or complicated operations required in [the] rolling circle amplification method,” the scientists noted.

Investigators from South China Normal University have introduced a new label-free approach for the detection of microRNAs.

The system is based on a novel graphene oxide/intercalating dye-based fluorescent hairpin probe and an isothermal polymerization reaction, according to a paper in Chemistry. Using miR-21 as a target for proof of concept, the team hybridized the miRNA with the probe, causing a conformation change and activating the polymerization — the first step of the signal amplification.

After incubation with the graphene oxide/intercalating dye, “the formed complex of DNA interacted with the high-affinity dye and then detached from the surface of the [graphene oxide], a process that was accompanied by distinguishable fluorescence recovery,” the researchers wrote. “Further signal enhancement has been accomplished by a mass of intercalating dye inserting into the minor groove of the long duplex replication product.”

The “efficient and multiple amplification steps” of the approach triggered a “substantial enhancement” of sensitivity, enabling the rapid and selective detection of miR-21 at attomole levels.

“Our method has completely avoided laborious chemical modifications,” compared with conventional miRNA sensors that rely on sophisticated labeling steps, the research team wrote. And while the sensitivity of the method is not a “prominent advantage” over certain multiple enzyme-based exponential amplification methods, it could reduce the cost involved and simplify experimental operations.

“This cost-effective [graphene oxide]-based fluorescence amplification assay could, in principle, be extended to the detection of nucleic acids and may promote the application of graphene materials in biochemical studies and clinical diagnosis,” they added.