Quickly, 10 ng of total RNA was used per person RT response; 0.67 mL from the resultant cDNA was found in 10 L qPCR reactions. that are enriched in EVs that may be functionally moved between cells, supporting a regulated form of cell-cell communication. Graphical Abstract INTRODUCTION The majority of the human genome is transcribed into RNA, but only ~2%C3% encodes protein (Hangauer et al., 2013). Only a small fraction of noncoding RNA transcripts have been characterized, but they appear to play important regulatory roles in multiple biological contexts (Kopp and Mendell, 2018; Wu et al., 2017). Recently, numerous studies have demonstrated the presence of distinct types of extracellular RNA (exRNA) in diverse biological fluids, adding yet another surprise to the overall role of RNA in gene expression (Colombo et al., 2014; Mateescu et al., 2017; Tkach and Thry, 2016). Because extracellular fluids display abundant ribonuclease activity, exRNA must be protected from degradation in protein complexes (Arroyo et al., 2011; Turchinovich et al., 2011), lipid complexes (Tabet et al., 2014; Vickers et al., 2011), or extracellular vesicles (EVs) (Ratajczak et al., 2006; Skog et al., 2008; Valadi et al., 2007). EVs refer to membrane limited nanovesicles including exosomes, microvesicles, and other secreted vesicles (Raposo and Stoorvogel, 2013). Each class of vesicle is unique in its origin and/or size and thus differs in its composition of lipid, protein, RNA, and potential DNA cargo (Colombo et al., 2014; Mateescu et al., 2017). EVs are released by all cell types and can serve as vehicles for transport of protein and RNA cargo between cells, representing a potential mechanism for intercellular communication (Ratajczak et al., 2006; Skog et al., 2008; Valadi et al., 2007). Local and systemic cargo transfer via EVs has been associated with tumor microenvironment interactions, aggressiveness, and metastasis (Becker et al., 2016; Kalluri, 2016; Shurtleff et al., 2018). This potentially allows secretion of proteins and RNAs that could inhibit local Treprostinil sodium growth and simultaneously educate distant tissues for metastasis (Peinado et al., 2012). Circulating RNAs encased in vesicles or protein complexes are often altered in cancer and bear tumor-type-specific signatures, making them attractive candidates as hN-CoR clinical biomarkers for disease diagnosis and prognosis (Quinn et al., 2015). Many exRNA studies have focused on miRNAs because they are well characterized, small, relatively stable, and well annotated (Cha et al., 2015; Mittelbrunn et al., 2011; Valadi et al., 2007; Vickers et al., 2011). However, the diversity of exRNA is extensive and microRNAs (miRNAs) are not the most abundant class of RNA found in EVs (Fritz et al., 2016; Mateescu et al., 2017). Analysis of cellular versus exRNA has repeatedly demonstrated selective biogenesis, export, and/or stability of specific RNAs (Cha et al., 2015; Dou et al., 2016; Kosaka et Treprostinil sodium al., 2010; Santangelo et al., 2016; Skog et al., 2008; Squadrito Treprostinil sodium et al., 2014; Valadi et al., 2007; Villarroya-Beltri et al., 2013; Wei et al., 2017). Elucidation of the mechanisms for selective sorting of cargo into EVs is critical to understanding extracellular signaling by RNA. In our ongoing efforts to understand the biological and pathological role of exRNAs regulated by oncogenic signaling, we utilized three isogenic colorectal cancer (CRC) cell lines that differ only in the mutational status of the gene (Shirasawa et al., 1993). mutations occur in ~34%C45% of colon cancers (Wong and Cunningham, 2008). The parental DLD-1 cell line contains both WT and G13D mutant alleles, while the isogenically matched derivative cell lines contain only one mutant allele (DKO-1) or one WT allele (DKs-8) (Shirasawa et al., 1993). We previously showed that EVs from mutant CRC cells can be transferred to WT cells to induce cell growth, migration, and invasiveness (Demory Beckler et al., 2013; Higginbotham et al., 2011). Additionally, we found that the miRNA profiles of EVs from all three cell lines are distinct from the parental cells and segregate depending on KRAS status and that specific miRNAs can be functionally transferred from mutant KRAS cells to WT cells (Cha et al., 2015). We also found that specific intracellular oncogenic signaling events can regulate trafficking of miRNAs through Treprostinil sodium phosphorylation of Argonaute (AGO) proteins (McKenzie et al., 2016). More recently, we identified a global downregulation of circular RNAs (circRNAs) in mutant.