The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments. Keywords: extracellular vesicle; exosome; microvesicle; standardization; isolation (Published: 27 May 2013) Citation: Journal of Extracellular Vesicles 2013, 2 : 20360 - http://dx.doi.org/10.3402/jev.v2i0.20360

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Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

Introduction : microRNA (miRNA) are small non-coding RNA species that are transcriptionally processed in the host cell and released extracellularly into the bloodstream. Normally involved in post-transcriptional gene silencing, the deregulation of miRNA has been shown to influence pathogenesis of a number of diseases. Background : Next-generation deep sequencing (NGS) has provided the ability to profile miRNA in biological fluids making this approach a viable screening tool to detect miRNA biomarkers. However, collection and handling procedures of blood needs to be greatly improved for miRNA analysis in order to reliably detect differences between healthy and disease patients. Furthermore, ribonucleases present in blood can degrade RNA upon collection rendering extracellular miRNA at risk of degradation. These factors have consequently decreased sensitivity and specificity of miRNA biomarker assays. Method : Here, we use NGS to profile miRNA in various blood components and identify differences in profiles within peripheral blood compared to cell-free plasma or serum and extracellular vesicles known as exosomes. We also analyse and compare the miRNA content in exosomes prepared by ultracentrifugation methods and commercial exosome isolation kits including treating samples with RNaseA. Conclusion : This study demonstrates that exosomal RNA is protected by RNaseA treatment and that exosomes provide a consistent source of miRNA for disease biomarker detection. Keywords: microRNA; exosomes; serum; plasma; deep sequencing (Published: 26 March 2014) Citation: Journal of Extracellular Vesicles 2014, 3 : 23743 - http://dx.doi.org/10.3402/jev.v3.23743 To access the supplementary material to this article, please see Supplementary files under Article Tools online.

https://www.tandfonline.com/doi/pdf/10.3402/jev.v3.23743

Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood

Diabetes mellitus is characterized by insulin secretion from pancreatic β cells that is insufficient to maintain blood glucose homeostasis. Autoimmune destruction of β cells results in type 1 diabetes mellitus, whereas conditions that reduce insulin sensitivity and negatively affect β-cell activities result in type 2 diabetes mellitus. Without proper management, patients with diabetes mellitus develop serious complications that reduce their quality of life and life expectancy. Biomarkers for early detection of the disease and identification of individuals at risk of developing complications would greatly improve the care of these patients. Small non-coding RNAs called microRNAs (miRNAs) control gene expression and participate in many physiopathological processes. Hundreds of miRNAs are actively or passively released in the circulation and can be used to evaluate health status and disease progression. Both type 1 diabetes mellitus and type 2 diabetes mellitus are associated with distinct modifications in the profile of miRNAs in the blood, which are sometimes detectable several years before the disease manifests. Moreover, circulating levels of certain miRNAs seem to be predictive of long-term complications. Technical and scientific obstacles still exist that need to be overcome, but circulating miRNAs might soon become part of the diagnostic arsenal to identify individuals at risk of developing diabetes mellitus and its devastating complications.

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Circulating microRNAs as novel biomarkers for diabetes mellitus

Diabetic nephropathy is a significant cause of chronic kidney disease and end-stage renal failure globally Much research has been conducted in both basic science and clinical therapeutics, which has enhanced understanding of the pathophysiology of diabetic nephropathy and expanded the potential therapies available This review will examine the current concepts of diabetic nephropathy management in the context of some of the basic science and pathophysiology aspects relevant to the approaches taken in novel, investigative treatment strategies

/pdf/diabetic-nephropathy-complications-and-treatment-arovwpix56.pdf

Diabetic nephropathy - complications and treatment.

MicroRNAs (miRNAs) have emerged as important regulators in the post-transcriptional control of gene expression. The discovery of their presence not only in tissues but also in extratissular fluids, including blood, urine and cerebro-spinal fluid, together with their changes in expression in various pathological conditions, has implicated these extracellular miRNAs as informative biomarkers of disease. However, exploiting miRNAs in this capacity requires methodological rigour. Here, we report several key procedural aspects of miRNA isolation from plasma and serum, as exemplified by research in cardiovascular and pulmonary diseases. We also highlight the advantages and disadvantages of various profiling methods to determine the expression levels of plasma- and serum-derived miRNAs. Attention to such methodological details is critical, as circulating miRNAs become diagnostic tools for various human diseases.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/jcmm.12236

Methodological challenges in utilizing miRNAs as circulating biomarkers.

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate various biological processes, primarily through interaction with messenger RNAs. The levels of specific, circulating miRNAs in blood have been shown to associate with various pathological conditions including cancers. These miRNAs have great potential as biomarkers for various pathophysiological conditions. In this study we focused on different sample types' effects on the spectrum of circulating miRNA in blood. Using serum and corresponding plasma samples from the same individuals, we observed higher miRNA concentrations in serum samples compared to the corresponding plasma samples. The difference between serum and plasma miRNA concentration showed some associations with miRNA from platelets, which may indicate that the coagulation process may affect the spectrum of extracellular miRNA in blood. Several miRNAs also showed platform dependent variations in measurements. Our results suggest that there are a number of factors that might affect the measurement of circulating miRNA concentration. Caution must be taken when comparing miRNA data generated from different sample types or measurement platforms.

/pdf/comparing-the-microrna-spectrum-between-serum-and-plasma-403rvycrvm.pdf

Comparing the MicroRNA spectrum between serum and plasma.

Background
Patients with Type 1 Diabetes (T1D) are particularly vulnerable to development of Diabetic nephropathy (DN) leading to End Stage Renal Disease. Hence a better understanding of the factors affecting kidney disease progression in T1D is urgently needed. In recent years microRNAs have emerged as important post-transcriptional regulators of gene expression in many different health conditions. We hypothesized that urinary microRNA profile of patients will differ in the different stages of diabetic renal disease.

/pdf/urinary-microrna-profiling-in-the-nephropathy-of-type-1-u0bwi0mk3t.pdf

Urinary MicroRNA Profiling in the Nephropathy of Type 1 Diabetes

A second grant from the NIH was incorrectly omitted from the Funding statement. The grant number is {"type":"entrez-nucleotide","attrs":{"text":"DK047874","term_id":"187373933","term_text":"DK047874"}}DK047874.

Sara McClarty

Papers

Comparing the MicroRNA spectrum between serum and plasma.

Urinary MicroRNA Profiling in the Nephropathy of Type 1 Diabetes

Correction: Urinary MicroRNA Profiling in the Nephropathy of Type 1 Diabetes