The Small RNA Repertoire of Small Extracellular Vesicles Isolated From Donor Kidney Preservation Fluid Provides a Source for Biomarker Discovery for Organ Quality and Posttransplantation Graft Function.

Unlabelled: Delayed graft function (DGF) after kidney transplantation is negatively associated with long-term graft function and survival. Kidney function after transplantation depends on multiple factors, both donor- and recipient-associated. Prediction of posttransplantation graft function would allow timely intervention to optimize patient care and survival.

Isolation of Extracellular Vesicles: General Methodologies and Latest Trends.

Background: Extracellular vesicles (EVs) play an essential role in the communication between cells and transport of diagnostically significant molecules. A wide diversity of approaches utilizing different biochemical properties of EVs and a lack of accepted protocols make data interpretation very challenging.

Scope Of Review: This review consolidates the data on the classical and state-of-the-art methods for isolation of EVs, including exosomes, highlighting the advantages and disadvantages of each method.

Strategies for isolation of exosomes.

Exosomes are tiny vesicles (diameter 30-150 nm) secreted by cells in culture and found in all body fluids. These vesicles, loaded with unique RNA and protein cargos, have many biological functions, of which only a small fraction is currently understood-for example, they participate in cell-to-cell communication and signaling within the human body. The spectrum of current scientific interest in exosomes is wide and ranges from understanding their functions and pathways to using them in diagnostics, as biomarkers, and in the development of therapeutics.

An optimized procedure for exosome isolation and analysis using serum samples: Application to cancer biomarker discovery.

Exosomes are RNA and protein-containing nanovesicles secreted by all cell types and found in abundance in body fluids, including blood, urine and cerebrospinal fluid. These vesicles seem to be a perfect source of biomarkers, as their cargo largely reflects the content of parental cells, and exosomes originating from all organs can be obtained from circulation through minimally invasive or non-invasive means. Here we describe an optimized procedure for exosome isolation and analysis using clinical samples, starting from quick and robust extraction of exosomes with Total exosome isolation reagent, then isolation of RNA followed by qRT-PCR.

Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers.

Exosomes are tiny vesicles (30-150 nm) constantly secreted by all healthy and abnormal cells, and found in abundance in all body fluids. These vesicles, loaded with unique RNA and protein cargo, have a wide range of biological functions, including cell-to-cell communication and signalling. As such, exosomes hold tremendous potential as biomarkers and could lead to the development of minimally invasive diagnostics and next generation therapies within the next few years.

Enhanced cellular uptake and gene silencing activity of siRNA molecules mediated by chitosan-derivative nanocomplexes.

The RNA interference (RNAi) constitutes a conservative mechanism in eukaryotic cells that induces silencing of target genes. In mammalians, the RNAi is triggered by siRNA (small interfering RNA) molecules. Due to its potential in silencing specific genes, the siRNA has been considered a potential alternative for the treatment of genetic and acquired diseases.

Discovery of a dicer-independent, cell-type dependent alternate targeting sequence generator: implications in gene silencing & pooled RNAi screens.

There is an acceptance that plasmid-based delivery of interfering RNA always generates the intended targeting sequences in cells, making it as specific as its synthetic counterpart. However, recent studies have reported on cellular inefficiencies of the former, especially in light of emerging gene discordance at inter-screen level and across formats. Focusing primarily on the TRC plasmid-based shRNA hairpins, we reasoned that alleged specificities were perhaps compromised due to altered processing; resulting in a multitude of random interfering sequences.

Expression of B-cell surface antigens in subpopulations of exosomes released from B-cell lymphoma cells.

Purpose: Exosomes are small (30- to 100-nm) vesicles secreted by all cell types in culture and found in most body fluids. A mean of 1 mL of blood serum, derived from healthy donors, contains approximately 10(12) exosomes. Depending on the disease, the number of exosomes can fluctuate.

miRNA-128 suppresses prostate cancer by inhibiting BMI-1 to inhibit tumor-initiating cells.

microRNA-128 (miR128) is reduced in prostate cancer relative to normal/benign prostate tissues, but causal roles are obscure. Here we show that exogenously introduced miR128 suppresses tumor regeneration in multiple prostate cancer xenograft models. Cancer stem-like cell (CSC)-associated properties were blocked, including holoclone and sphere formation as well as clonogenic survival.

The complete exosome workflow solution: from isolation to characterization of RNA cargo.

Exosomes are small (30-150 nm) vesicles containing unique RNA and protein cargo, secreted by all cell types in culture. They are also found in abundance in body fluids including blood, saliva, and urine. At the moment, the mechanism of exosome formation, the makeup of the cargo, biological pathways, and resulting functions are incompletely understood.

Methods for the extraction and RNA profiling of exosomes.

Aim: To develop protocols for isolation of exosomes and characterization of their RNA content.

Methods: Exosomes were extracted from HeLa cell culture media and human blood serum using the Total exosome isolation (from cell culture media) reagent, and Total exosome isolation (from serum) reagent respectively. Identity and purity of the exosomes was confirmed by Nanosight(®) analysis, electron microscopy, and Western blots for CD63 marker.

Control of cholesterol metabolism and plasma high-density lipoprotein levels by microRNA-144.

Rationale: Foam cell formation because of excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis, the major cause of morbidity and mortality in Western societies. Liver X nuclear receptors (LXRs) regulate the expression of the adenosine triphosphate-binding cassette (ABC) transporters, including adenosine triphosphate-binding cassette transporter A1 (ABCA1) and adenosine triphosphate-binding cassette transporter G1 (ABCG1). ABCA1 and ABCG1 facilitate the efflux of cholesterol from macrophages and regulate high-density lipoprotein (HDL) biogenesis.

Intravital fluorescence imaging of small interfering RNA-mediated gene repression in a dual reporter melanoma xenograft model.

Development of RNA interference (RNAi)-based therapeutics has been hampered by the lack of effective and efficient means of delivery. Reliable model systems for screening and optimizing delivery of RNAi-based agents in vivo are crucial for preclinical research aimed at advancing nucleic acid-based therapies. We describe here a dual fluorescent reporter xenograft melanoma model prepared by intradermal injection of human A375 melanoma cells expressing tandem tomato fluorescent protein (tdTFP) containing a small interfering RNA (siRNA) target site as well as enhanced green fluorescent protein (EGFP), which is used as a normalization control.

Formulation approaches to short interfering RNA and MicroRNA: challenges and implications.

RNA interference has emerged as a potentially powerful tool in the treatment of genetic and acquired diseases by delivering short interfering RNA (siRNA) or microRNA (miRNA) to target genes, resulting in their silencing. However, many physicochemical and biological barriers have to be overcome to obtain efficient in vivo delivery of siRNA and miRNA molecules to the organ/tissue of interest, thereby enabling their effective clinical therapy. This review discusses the challenges associated with the use of siRNA and miRNA and describes the nonviral delivery strategies used in overcoming these barriers.

Distinct microRNA expression profiles in prostate cancer stem/progenitor cells and tumor-suppressive functions of let-7.

MiRNAs regulate cancer cells, but their potential effects on cancer stem/progenitor cells are still being explored. In this study, we used quantitative real-time-PCR to define miRNA expression patterns in various stem/progenitor cell populations in prostate cancer, including CD44+, CD133+, integrin α2β1+, and side population cells. We identified distinct and common patterns in these different tumorigenic cell subsets.

Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials.

Background: Cells continuously secrete a large number of microvesicles, macromolecular complexes, and small molecules into the extracellular space. Of the secreted microvesicles, the nanoparticles called exosomes are currently undergoing intense scrutiny. These are small vesicles (30-120 nm) containing nucleic acid and protein, perceived to be carriers of this cargo between diverse locations in the body.

Aquaporins mediate the chemoresistance of human melanoma cells to arsenite.

The integral membrane channel protein aquaporin (AQP) is aberrantly expressed with oncogenic characteristics in various human cancers. In this study, we analyzed the expression pattern of all subtypes of AQPs, and found that 8 out of 13 AQPs expressed in melanoma cells. To understand the role of aberrant expression of AQP in this disease, we over-expressed AQP3 and AQP9 in human melanoma WM266.

Optimization of transfection conditions and analysis of siRNA potency using real-time PCR.

RNA interference (RNAi) is a mechanism by which the introduction of small interfering RNAs (siRNAs) into cultured cells causes degradation of the complementary mRNA. Applications of RNAi include gene function analysis, pathway analysis, and target validation. While RNAi experiments have become common practice in research labs, multiple factors can influence the extent of siRNA-induced knockdown (and thus biological outcome).

Quantification of siRNAs in vitro and in vivo.

RNA interference (RNAi) is a regulatory mechanism of eukaryotic cells that uses small interfering RNAs (siRNA) to direct homology-dependent control of gene activity. Applications of RNAi include functional genomics, in vivo target validation, and gene-specific medicines. A key to in vivo application of siRNA is the advancement of efficient delivery to organs, tissues, or cell types of interest.

Efficient gene silencing in lungs and liver using imidazole-modified chitosan as a nanocarrier for small interfering RNA.

Despite high specificity and potency, small interfering RNA (siRNA)-based therapeutics have been limited by their poor biostability and intracellular penetration. Thus, effective nanocarriers that can protect and efficiently deliver siRNA to target cells in vivo are needed. Here we report on the efficiency of imidazole-modified chitosan (chitosan-imidazole-4-acetic acid [IAA])-siRNA nanoparticles to mediate gene silencing after administration via either intravenous (i.

Locked nucleic acid (LNA)-modified dinucleotide mRNA cap analogue: synthesis, enzymatic incorporation, and utilization.

There has been considerable therapeutic interest in the development of human vaccines against cancers and infectious diseases such as HIV and biowarfare agents by using transfected mRNAs for antigenic proteins of interest. The highest expression levels of these proteins are obtained when the transfected mRNA contains 5'-capped ends. In the present study, the locked nucleic acid (LNA)-modified cap analogue 3, m(7(LNA))G[5']ppp[5']G, has been synthesized and its biological properties were examined.

Stem-loop RT-PCR quantification of siRNAs in vitro and in vivo.

RNA interference (RNAi) is a mechanism in which the introduction of small interfering RNAs (siRNAs) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. Applications of RNAi include gene function and pathway analysis, target identification and validation, and therapeutics. There is a need to develop reliable and easy-to-use assays to evaluate siRNA delivery efficiency and distribution, study pathways, and stability of siRNAs in cells (posttransfection) and in animals (postinjection).

Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos.

We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5'- and 3'-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA.

Stability study of unmodified siRNA and relevance to clinical use.

RNA interference offers enormous potential to develop therapeutic agents for a variety of diseases. To assess the stability of siRNAs under conditions relevant to clinical use with particular emphasis on topical delivery considerations, a study of three different unmodified siRNAs was performed. The results indicate that neither repeated freeze/thaw cycles, extended incubations (over 1 year at 21 degrees C), nor shorter incubations at high temperatures (up to 95 degrees C) have any effect on siRNA integrity as measured by nondenaturing polyacrylamide gel electrophoresis and functional activity assays.

Synthesis and application of a new 2',3'-isopropylidene guanosine substituted cap analog.

The synthesis and biological evaluation of a new cap analog, which is modified at the C2' and C3' positions of N(7)-methylguanosine is reported. The new cap analog, P(1)-2',3'-isopropylidene, 7-methylguanosine-5'P(3)-guanosine-5'triphosphate was assayed with respect to its effects on efficiency of incorporation into RNAs during in vitro transcription, and intracellular stability and translational activity of its 5'-capped mRNAs, upon transfection into HeLa cells. The intracellular stability of 5'-capped and uncapped full length test mRNAs was measured by using a real-time RT-PCR assay.