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Tools & Techniques: siRNA Delivery to Endothelia; miRNA Analysis in Non-Model Animals; and More


With an eye toward applying RNAi to the treatment of disorders associated with injured endothelial cells, a team of researchers from Huazhong University of Science and Technology has reported on novel nanoparticles capable of delivering siRNAs into such cells in the brain.

Brain microvascular endothelial cells, or BMECs, act as a barrier to pathogens in the brain, and injury to these cells is associated with the development of a variety of diseases including stroke and atherosclerosis.

In order to explore the targeted delivery of siRNAs to BMECs, the research team focused on the plasma membrane glycoprotein tissue factor, or TF, which initiates blood coagulation and is overexpressed by injured BMECs, according to a paper in PLoS One.

They created nanoparticles composed of poly(lactic-co-glycolic acid), a biodegradable and biocompatible polymer, and decorated them with the fusion protein EGFP-EGF1, which can be used to target TF and, therefore, cells that express it. The nanoparticles were then loaded with siRNAs designed to inhibit the protein, and administered to injured BMECs in vitro.

The researchers found that they were able to trigger an RNAi effect against TF using the targeted nanoparticles more efficiently than with untargeted nanoparticles, they wrote in the paper. “Our findings also show that the TF siRNA-loaded [targeted nanoparticles] had minimal toxicity, with almost 96 percent of the cells viable 24 [hours] after transfection while Lipofectamine-based transfections resulted in only 75 percent of the cells.”

Seeing potential for the approach based on these findings, the team aims to test the nanoparticles’ use in vivo.

Also aiming to overcome delivery challenges facing RNAi, a research group led by investigators from the University of Iowa have published details of a strategy to introduce siRNAs, Dicer-substrates, and microRNA oligos into airway epithelia.

“Through gain- and loss-of-function experiments, investigators have the opportunity to investigate the role of specific gene products in biologic processes,” they wrote in the American Journal of Physiology — Lung Cellular and Molecular Physiology. “However, the pseudostratified epithelium presents significant barriers to the delivery of nucleic acid-based reagents such as plasmids, double-stranded oligonucleotides, and single-stranded oligonucleotides.”

Additionally, airway secretions, host defense mechanism, and the mucosal surface of the airways and alveoli further hinders the effective delivery of reagents such as siRNAs.

With this in mind, the scientists developed a “simple” approach called reverse transfection wherein reagent complexes are added to cells at the time of seeding, thereby exploiting cells’ susceptibility to transfection during plating.

“In contrast to standard transfection where transfection complexes are added to pre-plated cells, reverse transfection is performed by adding the cells and the transfection complexes into the wells essentially at the same time,” they wrote.

The team demonstrated that the technique is effective in primary human and porcine airway epithelial cells, Calu-3 cells, T84 cells, and CFBE cells without impacting cell viability. And while not formally tested, they noted that they expect it to also work in mouse epithelial cells and other difficult-to-transfect primary cells and cell lines.

“This method provides a novel avenue for researchers to circumvent the barrier properties of polarized, pseudostratified primary airway epithelia,” the researchers concluded. “By transfecting poorly differentiated cells, investigators can conduct experiments and address questions that apply to polarized, pseudostratifiedcells, while maintaining knockdown of targeted genes.”

Although tools exist to predict miRNA precursors and targets, the majority of these are geared for use only with model organisms. However, by combining different next-generation sequencing technologies, a research group from the University of Nordland has been able to identify a number of pre-miRNAs and predict various miRNA targets for the Atlantic halibut.

In previous studies, the investigators generated miRNA libraries from various stages of the fish’s development, and the immature adult male and female brain and gonad, according to a paper appearing in PLoS One. They also have shown differential expression of various miRNAs during the halibut’s major developmental transitions and its sexually dimorphic expression during sexual development.

In their new study, the scientists sequenced the animal’s genomic DNA and the small transcriptome using Roche 454 pyrosequencing and SOLiD next-generation sequencing, respectively. Pre-miRNAs that were identified were further validated using reverse-transcription PCR, while miRNA targets were identified with two different target prediction tools using sequences from public databases.

Some of miRNA targets were identified using rapid amplification of cDNA ends PCR, and miRNA binding sites were validated with a luciferase assay.

With this combination of technologies, the researchers were able to obtain more than 1.3 million sequence reads from the 454 genomic DNA sequencing and 92 million from the SOLiD small RNA sequencing. They identified 34 known pre-miRNAs and nine novel ones, and predicted a variety of miRNA target genes involved in different biological pathways, one of which was validated using the luciferase assay.

The methodology used in the study shows that next-generation sequencing is effective in “deciphering and characterizing pre-miRNAs and miRNA targets in animal species with limited genomic resources,” according to the paper. “The use of pathway models and other information from model species provide the possibility for functional annotation of genomic elements in non-model species,” while also allowing extensive comparative studies.

Despite the availability of a number of techniques for miRNA sequencing and profiling, achieving high sensitivity, single-base specificity, and a broad dynamic range simultaneously remains a challenge. To overcome this limitation, researchers from the University of Houston have developed a new label-free miRNA detection method with potential for diagnostic applications.

The approach is based on exchange-induced remnant magnetization, or EXIRM, which takes advantage of the exchange reaction between target miRNA and magnetically labeled RNA with one mismatched base during competitive binding with the complementary sequence of the target miRNA, according to a paper in Chemical Communications. During such reactions, the decrease in magnetization quantitatively represents the target miRNA.

The researchers state that the detection limit of the technique reaches the zeptomole regime, with no amplification or washing procedures requires, which makes it “suitable for precise miRNA profiling to aid in early diagnosis of cancers.”