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.

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.

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.

An arrayed RNA interference genome-wide screen identifies candidate genes involved in the MicroRNA 21 biogenesis pathway.

MicroRNAs (miRNAs) are evolutionary conserved noncoding molecules that regulate gene expression. They influence a number of diverse biological functions, such as development and differentiation. However, their dysregulation has been shown to be associated with disease states, such as cancer.