Pre-diagnosis urine exosomal RNA (ExoDx EPI score) is associated with post-prostatectomy pathology outcome.

PURPOSE: ExoDx Prostate IntelliScore (EPI) is a non-invasive urine exosome RNA-based test for risk assessment of high-grade prostate cancer. We evaluated the association of pre-biopsy test results with post-radical prostatectomy (RP) outcomes to understand the potential utility of EPI to inform invasive treatment vs active surveillance (AS) decisions. METHODS: Urine samples were collected from 2066 men scheduled for initial biopsy with PSA between 2 and 10 ng/mL, no history of prostate cancer, and ≥ 50 years across multiple clinical studies. 310 men proceeded to RP, of which 111 patients had Gleason group grade 1 (GG1) at biopsy and would have been potential candidates for AS. We compared pre-biopsy urine scores with ERSPC and PCPT multivariate risk calculator scores for men with GG1 at biopsy to post-RP pathology. RESULTS: Urine EPI scores were significantly lower in men with GG1 at biopsy than in men with > GG1 (p = 0.04), while there were no differences in multivariate risk scores used in standard clinical practice (p > 0.05). Further, EPI scores were significantly lower in men with GG1 at biopsy who remained GG1 post-RP compared to men upgraded to ≥ GG3 post-RP (p < 0.001). In contrast, none of the multiparametric risk calculators showed significant differences (p > 0.05). Men with GG1 at biopsy and EPI score < 15.6 had zero rate of upgrading to ≥ GG3 post-RP compared to 16.0% for EPI scores ≥ 15.6. CONCLUSIONS: The EPI urine biomarker outperformed the multivariate risk calculators in a homogenous risk group of pre-biopsy men. The EPI score was associated with low-risk pathology post-RP, with potential implications on informing AS decisions. TRIAL REGISTRATION: NCT02702856, NCT03031418, NCT03235687, NCT04720599.

Discovery and Validation of a Urinary Exosome mRNA Signature for the Diagnosis of Human Kidney Transplant Rejection.

BACKGROUND: Developing a noninvasive clinical test to accurately diagnose kidney allograft rejection is critical to improve allograft outcomes. Urinary exosomes, tiny vesicles released into the urine that carry parent cells' proteins and nucleic acids, reflect the biologic function of the parent cells within the kidney, including immune cells. Their stability in urine makes them a potentially powerful tool for liquid biopsy and a noninvasive diagnostic biomarker for kidney-transplant rejection. METHODS: Using 192 of 220 urine samples with matched biopsy samples from 175 patients who underwent a clinically indicated kidney-transplant biopsy, we isolated urinary exosomal mRNAs and developed rejection signatures on the basis of differential gene expression. We used crossvalidation to assess the performance of the signatures on multiple data subsets. RESULTS: An exosomal mRNA signature discriminated between biopsy samples from patients with all-cause rejection and those with no rejection, yielding an area under the curve (AUC) of 0.93 (95% CI, 0.87 to 0.98), which is significantly better than the current standard of care (increase in eGFR AUC of 0.57; 95% CI, 0.49 to 0.65). The exosome-based signature's negative predictive value was 93.3% and its positive predictive value was 86.2%. Using the same approach, we identified an additional gene signature that discriminated patients with T cell-mediated rejection from those with antibody-mediated rejection (with an AUC of 0.87; 95% CI, 0.76 to 0.97). This signature's negative predictive value was 90.6% and its positive predictive value was 77.8%. CONCLUSIONS: Our findings show that mRNA signatures derived from urinary exosomes represent a powerful and noninvasive tool to screen for kidney allograft rejection. This finding has the potential to assist clinicians in therapeutic decision making.

A urine-based Exosomal gene expression test stratifies risk of high-grade prostate Cancer in men with prior negative prostate biopsy undergoing repeat biopsy.

BACKGROUND: Initial prostate biopsy often fails to identify prostate cancer resulting in patient anxiety, especially when clinical features such as prostate specific antigen (PSA) remain elevated, leading to the need for repeat biopsies. Prostate biomarker tests, such as the ExoDx™ Prostate (IntelliScore), or EPI test, have been shown to provide individualized risk assessment of clinically significant prostate cancer at initial biopsy; however, the performance in the repeat biopsy setting is not well established. METHODS: As part of a previous prospective clinical validation study evaluating the performance of the EPI test, we collected first-catch, non-DRE urine samples across 22 sites from men with at least one prior negative biopsy scheduled to undergo a repeat prostate biopsy to rule out prostate cancer. All men were 50 years or older with a PSA 2-10 ng/mL. Exosomal mRNA was extracted and expression of three genomic markers, PCA3, ERG and SPDEF was measured. The resulting EPI score was correlated with biopsy results. RESULTS: 229 men with a prior negative biopsy underwent repeat biopsies. ExoDx Prostate demonstrated good performance ruling out high-grade (Grade group 2, GG2, or higher) prostate cancer (HGPCa) using the previously validated 15.6 cut point in the initial biopsy setting. The EPI test yielded an NPV of 92% independent of other clinical features and would have avoided 26% of unnecessary biopsies while missing only five patients with HGPCa (2.1%). Furthermore, the EPI test provided additional information at a cut-point of 20 and 29.6 with an NPV of 94%, potentially delaying 35 and 61% of unnecessary biopsies, respectively. AUC curves and Net Health Benefit Analyses demonstrated superior performance of ExoDx Prostate over PSA and clinical only risk calculators, i.e. ERSPC. CONCLUSIONS: The EPI test provided good performance using the 15.6 cut-point for ruling out HGPCa / GG2 or higher in men undergoing a repeat prostate biopsy with a PSA of 2-10 ng/ml. Furthermore, the test utilizes gene expression data independent of clinical features to predict the likelihood of HGPCa / GG2 on a subsequent needle biopsy.

Current methods for the isolation of extracellular vesicles.

Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter.

Blood platelets contain tumor-derived RNA biomarkers.

Diagnostic platforms providing biomarkers that are highly predictive for diagnosing, monitoring, and stratifying cancer patients are key instruments in the development of personalized medicine. We demonstrate that tumor cells transfer (mutant) RNA into blood platelets in vitro and in vivo, and show that blood platelets isolated from glioma and prostate cancer patients contain the cancer-associated RNA biomarkers EGFRvIII and PCA3, respectively. In addition, gene-expression profiling revealed a distinct RNA signature in platelets from glioma patients compared with normal control subjects. Because platelets are easily accessible and isolated, they may form an attractive platform for the companion diagnostics of cancer.

Heparin blocks transfer of extracellular vesicles between donor and recipient cells.

Extracellular vesicles (EVs) have been implicated in tumorigenesis. Biomolecules which can block EV binding and uptake into recipient cells may be of therapeutic value as well as enhance understanding of EV biology. Here, we show that heparin interacts with uptake of tumor-derived as well as non-tumor-derived EVs into recipient cells. Incubation of glioma cell-derived EVs with heparin resulted in micron-sized structures observed by transmission electron microscopy, with EVs clearly visible within these structures. Inclusion of heparin greatly diminished transfer of labeled EVs from donor to recipient tumor cells. We also show a direct interaction between heparin and EVs using confocal microscopy. We found that the block in EV uptake was at the level of cell binding and not internalization. Finally, incubation of glioma-derived EVs containing EGFRvIII mRNA with heparin reduced transfer of this message to recipient cells. The effect of heparin on EVs uptake may provide a unique tool to study EV function. It may also foster research of heparin or its derivatives as a therapeutic for disease in which EVs play a role.

Microvesicle-associated AAV vector as a novel gene delivery system.

Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery.

Cerebrospinal fluid as a liquid biopsy for molecular characterization of brain metastasis in patients with non-small cell lung cancer.

OBJECTIVES: Non-small cell lung cancer (NSCLC) with brain metastases (BM) is a challenging clinical issue with poor prognosis. No data exist regarding extensive genetic analysis of cerebrospinal fluid (CSF) and its correlation to associated tumor compartments. MATERIALS AND METHODS: We designed a study across multiple NSCLC patients with matched material from four compartments; primary tumor, BM, plasma and CSF. We performed enrichment-based targeted next-generation sequencing analysis of ctDNA and exosomal RNA in CSF and plasma and compared the outcome with the solid tumor compartments. RESULTS: An average of 105 million reads per sample was generated with fractions of mapped reads exceeding 99% in all samples and with a mean coverage above 10,000x. We observed a high degree of overlap in variants between primary lung tumor and BM. Variants specific for the BM/CSF compartment included in-frame deletions in AR, FGF10 and TSC1 and missense mutations in HNF1a, CD79B, BCL2, MYC, TSC2, TET2, NRG1, MSH3, NOTCH3, VHL and EGFR. CONCLUSION: Our approach of combining ctDNA and exosomal RNA analyses in CSF presents a potential surrogate for BM biopsy. The specific variants that were only observed in the CNS compartments could serve as targets for individually tailored therapies in NSCLC patients with BM.

ExoDx prostate test as a predictor of outcomes of high-grade prostate cancer - an interim analysis.

BACKGROUND: Patient outcomes were assessed based on a pre-biopsy ExoDx Prostate (EPI) score at 2.5 years of the 5-year follow-up of ongoing prostate biopsy Decision Impact Trial of the ExoDx Prostate (IntelliScore). METHODS: Prospective, blinded, randomized, multisite clinical utility study was conducted from June 2017 to May 2018 (NCT03235687). Urine samples were collected from 1049 men (≥50 years old) with a PSA 2-10 ng/mL being considered for a prostate biopsy. Patients were randomized to EPI vs. standard of care (SOC). All had an EPI test, but only EPI arm received results during biopsy decision process. Clinical outcomes, time to biopsy and pathology were assessed among low (<15.6) or high (≥15.6) EPI scores. RESULTS: At 2.5 years, 833 patients had follow-up data. In the EPI arm, biopsy rates remained lower for low-risk EPI scores than high-risk EPI scores (44.6% vs 79.0%, p < 0.001), whereas biopsy rates were identical in SOC arm regardless of EPI score (59.6% vs 58.8%, p = 0.99). Also in the EPI arm, the average time from EPI testing to first biopsy was longer for low-risk EPI scores compared to high-risk EPI scores (216 vs. 69 days; p < 0.001). Similarly, the time to first biopsy was longer with EPI low-risk scores in EPI arm compared to EPI low-risk scores in SOC arm (216 vs 80 days; p < 0.001). At 2.5 years, patients with low-risk EPI scores from both arms had less HGPC than high-risk EPI score patients (7.9% vs 26.8%, p < 0.001) and the EPI arm found 21.8% more HGPC than the SOC arm. CONCLUSIONS: This follow-up analysis captures subsequent biopsy outcomes and demonstrates that men receiving EPI low-risk scores (<15.6) significantly defer the time to first biopsy and remain at a very low pathologic risk by 2.5-years after the initial study. The EPI test risk stratification identified low-risk patients that were not found with the SOC.

Longitudinal analysis of serum-derived extracellular vesicle RNA to monitor dacomitinib treatment response in EGFR-amplified recurrent glioblastoma patients.

Background: Glioblastoma (GBM) is a highly aggressive and invasive brain tumor associated with high patient mortality. A large fraction of GBM tumors have been identified as epidermal growth factor receptor (EGFR) amplified and ~50% also are EGFRvIII mutant positive. In a previously reported multicenter phase II study, we have described the response of recurrent GBM (rGBM) patients to dacomitinib, an EGFR tyrosine kinase inhibitor (TKI). As a continuation of that report, we leverage the tumor cargo-encapsulating extracellular vesicles (EVs) and explore their genetic composition as carriers of tumor biomarker. Methods: Serum samples were longitudinally collected from EGFR-amplified rGBM patients who clinically benefitted from dacomitinib therapy (responders) and those who did not (nonresponders), as well as from a healthy cohort of individuals. The serum EV transcriptome was evaluated to map the RNA biotype distribution and distinguish GBM disease. Results: Using long RNA sequencing, we show enriched detection of over 10 000 coding RNAs from serum EVs. The EV transcriptome yielded a unique signature that facilitates differentiation of GBM patients from healthy donors. Further analysis revealed genetic enrichment that enables stratification of responders from nonresponders prior to dacomitinib treatment as well as following administration. Conclusion: This study demonstrates that genetic composition analysis of serum EVs may aid in therapeutic stratification to identify patients with dacomitinib-responsive GBM.

Extrinsic and intrinsic preanalytical variables affecting liquid biopsy in cancer.

Liquid biopsy, through isolation and analysis of disease-specific analytes, has evolved as a promising tool for safe and minimally invasive diagnosis and monitoring of tumors. It also has tremendous utility as a companion diagnostic allowing detection of biomarkers in a range of cancers (lung, breast, colon, ovarian, brain). However, clinical implementation and validation remains a challenge. Among other stages of development, preanalytical variables are critical in influencing the downstream cellular and molecular analysis of different analytes. Although considerable progress has been made to address these challenges, a comprehensive assessment of the impact on diagnostic parameters and consensus on standardized and optimized protocols is still lacking. Here, we summarize and critically evaluate key variables in the preanalytical stage, including study population selection, choice of biofluid, sample handling and collection, processing, and storage. There is an unmet need to develop and implement comprehensive preanalytical guidelines on the optimal practices and methodologies.

The Liquid Biopsy Consortium: Challenges and opportunities for early cancer detection and monitoring.

The emerging field of liquid biopsy stands at the forefront of novel diagnostic strategies for cancer and other diseases. Liquid biopsy allows minimally invasive molecular characterization of cancers for diagnosis, patient stratification to therapy, and longitudinal monitoring. Liquid biopsy strategies include detection and monitoring of circulating tumor cells, cell-free DNA, and extracellular vesicles. In this review, we address the current understanding and the role of existing liquid-biopsy-based modalities in cancer diagnostics and monitoring. We specifically focus on the technical and clinical challenges associated with liquid biopsy and biomarker development being addressed by the Liquid Biopsy Consortium, established through the National Cancer Institute. The Liquid Biopsy Consortium has developed new methods/assays and validated existing methods/technologies to capture and characterize tumor-derived circulating cargo, as well as addressed existing challenges and provided recommendations for advancing biomarker assays.

RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls.

BACKGROUND: RNA from exosomes and other microvesicles contain transcripts of tumour origin. In this study we sought to identify biomarkers of glioblastoma multiforme in microvesicle RNA from serum of affected patients. METHODS: Microvesicle RNA from serum from patients with de-novo primary glioblastoma multiforme (N = 9) and normal controls (N = 7) were analyzed by microarray analysis. Samples were collected according to protocols approved by the Institutional Review Board. Differential expressions were validated by qRT-PCR in a separate set of samples (N = 10 in both groups). RESULTS: Expression profiles of microvesicle RNA correctly separated individuals in two groups by unsupervised clustering. The most significant differences pertained to down-regulated genes (121 genes > 2-fold down) in the glioblastoma multiforme patient microvesicle RNA, validated by qRT-PCR on several genes. Overall, yields of microvesicle RNA from patients was higher than from normal controls, but the additional RNA was primarily of size < 500 nt. Gene ontology of the down-regulated genes indicated these are coding for ribosomal proteins and genes related to ribosome production. CONCLUSIONS: Serum microvesicle RNA from patients with glioblastoma multiforme has significantly down-regulated levels of RNAs coding for ribosome production, compared to normal healthy controls, but a large overabundance of RNA of unknown origin with size < 500 nt.

Predicting high-grade prostate cancer at initial biopsy: clinical performance of the ExoDx (EPI) Prostate Intelliscore test in three independent prospective studies.

BACKGROUND: The ability to discriminate indolent from clinically significant prostate cancer (PC) at the initial biopsy remains a challenge. The ExoDx Prostate (IntelliScore) (EPI) test is a noninvasive liquid biopsy that quantifies three RNA targets in urine exosomes. The EPI test stratifies patients for risk of high-grade prostate cancer (HGPC; ≥ Grade Group 2 [GG] PC) in men ≥ 50 years with equivocal prostate-specific antigen (PSA) (2-10 ng/mL). Here, we present a pooled meta-analysis from three independent prospective-validation studies in men presenting for initial biopsy decision. METHODS: Pooled data from two prospective multi-site validation studies and the control arm of a clinical utility study were analyzed. Performance was evaluated using the area under the receiver-operating characteristic curve (AUC), negative predictive value (NPV), positive predictive value (PPV), sensitivity, and specificity for discriminating ≥ GG2 from GG1 and benign pathology. RESULTS: The combined cohort (n = 1212) of initial-biopsy subjects had a median age of 63 years and median PSA of 5.2 ng/mL. The EPI AUC (0.70) was superior to PSA (0.56), Prostate Cancer Prevention Trial Risk Calculator (PCPT-RC) (0.62), and The European Randomized Study of Screening for Prostate Cancer (ERSPC) (0.59), (all p-values <0.001) for discriminating GG2 from GG1 and benign histology. The validated cutoff of 15.6 would avoid 23% of all prostate biopsies and 30% of "unnecessary" (benign or Gleason 6/GG1) biopsies, with an NPV of 90%. CONCLUSIONS: EPI is a noninvasive, easy-to-use, urine exosome-RNA assay that has been validated across 3 independent prospective multicenter clinical trials with 1212 subjects. The test can discriminate high-grade (≥GG2) from low-grade (GG1) cancer and benign disease. EPI effectively guides the biopsy-decision process independent of PSA and other standard-of-care factors.

Exosome based analysis for Space Associated Neuro-Ocular Syndrome and health risks in space exploration.

Molecular profiling to characterize the effects of environmental exposures is important from the human health and performance as well as the occupational medicine perspective in space exploration. We have developed a novel exosome-based platform that allows profiling of biological processes in the body from a variety of body fluids. The technology is suitable for diagnostic applications as well as studying the pathophysiology of the Space Associated Neuro-Ocular Syndrome in astronauts and monitoring patients with chronically impaired cerebrospinal fluid drainage or elevated intracranial pressure. In this proof-of-concept, we demonstrate that: (a) exosomes from different biofluids contain a specific population of RNA transcripts; (b) urine collection hardware aboard the ISS is compatible with exosome gene expression technology; (c) cDNA libraries from exosomal RNA can be stored in dry form and at room temperature, representing an interesting option for the creation of longitudinal molecular catalogs that can be stored as a repository for retrospective analysis.

Validation of a CE-IVD, urine exosomal RNA expression assay for risk assessment of prostate cancer prior to biopsy.

Improved risk stratification of patients suspected of prostate cancer prior to biopsy continues to be an unmet clinical need. ExoDx Prostate (IntelliScore) "EPI" is a non-invasive urine test utilizing RNA from exosomes to provide a risk score that correlates with the likelihood of finding high grade prostate cancer at biopsy. Here, we present the results from a prospective clinical validation study of EPI-CE, a CE-marked in-vitro diagnostic (IVD) assay, specifically developed for use in European clinical laboratories. The study (NCT04720599) enrolled patients with ≥ 50 years, PSA 2-10 ng/mL, prior to MRI, who were scheduled for initial biopsy. First catch urine samples were collected from participants without prior digital rectal examination or prostate massage. Exosomal RNA was isolated and expression levels of three biomarkers ERG, PCA3 and SPDEF were analyzed according to the EPI-CE Instructions For Use. In the study cohort of N = 109 patients, EPI-CE was validated to have a Negative Predictive Value of 89%, a Sensitivity of 92% and a superior performance to two commonly used multiparametric risk calculators (PCPT and ERSPC) in both Receiver Operating Characteristics with a higher Area Under the Curve for EPI-CE 0.67 (95% CI 0.56-0.77) versus PCPT 0.59 (95% CI 0.47-0.71) and ERSPC 0.60 (95% CI 0.49-0.72) and higher Net Benefits analysis across a wide range of risk acceptance levels. This is the first clinical study reporting on the performance of EPI-CE. We demonstrate that EPI-CE provides information beyond standard clinical parameters and provides a better risk assessment prior to MRI, of patients suspected of prostate cancer, than the commonly used multiparametric risk calculators.

Brain tumor microvesicles: insights into intercellular communication in the nervous system.

Brain tumors are heterogeneous tumors composed of differentiated tumor cells that resemble various neural cells and a small number of multipotent cancer stem cells. These tumors modify normal cells in their environment to promote tumor growth, invasion and metastases by various ways. Recent publications show that glioblastoma cells release microvesicles that contain a select subset of cellular proteins and RNAs. These microvesicles are avidly taken up by normal cells in cell culture and can change the translational profile of these cells through delivery of tumor-derived mRNAs, which are translated into functional proteins. In addition to mRNA and proteins, microvesicles have been shown to contain microRNAs, non-coding RNAs and DNA. This commentary explores the recent advances in this novel intercellular communication route and discusses the potential physiological role of microvesicles in brain tumorigenesis.

A SARS-CoV-2 antigen rapid diagnostic test for resource limited settings.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 disease. RT-qPCR has been the primary method of diagnosis; however, the required infrastructure is lacking in many developing countries and the virus has remained a global challenge. More inexpensive and immediate test methods are required to facilitate local, regional, and national management strategies to re-open world economies. Here we have developed a SARS-CoV-2 antigen test in an inexpensive lateral flow format to generate a chromatographic result identifying the presence of the SARS-CoV-2 antigen, and thus an active infection, within a patient anterior nares swab sample. Our 15-min test requires no equipment or laboratory infrastructure to administer with a limit of detection of 2.0 × 102 TCID50/mL and 87.5% sensitivity, 100% specificity when tested against 40 known positive and 40 known negative patient samples established by a validated RT-qPCR test.

Microfluidic isolation and transcriptome analysis of serum microvesicles.

Microvesicles (exosomes) shed from both normal and cancerous cells may serve as means of intercellular communication. These microvesicles carry proteins, lipids and nucleic acids derived from the host cell. Their isolation and analysis from blood samples have the potential to provide information about state and progression of malignancy and should prove of great clinical importance as biomarkers for a variety of disease states. However, current protocols for isolation of microvesicles from blood require high-speed centrifugation and filtration, which are cumbersome and time consuming. In order to take full advantage of the potential of microvesicles as biomarkers for clinical applications, faster and simpler methods of isolation will be needed. In this paper, we present an easy and rapid microfluidic immunoaffinity method to isolate microvesicles from small volumes of both serum from blood samples and conditioned medium from cells in culture. RNA of high quality can be extracted from these microvesicles providing a source of information about the genetic status of tumors to serve as biomarkers for diagnosis and prognosis of cancer.

Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease.

Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery.