Abstract

Intact RNA from exosomes/microvesicles (collectively referred to as microvesicles) has sparked much interest as potential biomarkers for the non-invasive analysis of disease. Here we use the Illumina Genome Analyzer to determine the comprehensive array of nucleic acid reads present in urinary microvesicles. Extraneous nucleic acids were digested using RNase and DNase treatment and the microvesicle inner nucleic acid cargo was analyzed with and without DNase digestion to examine both DNA and RNA sequences contained in microvesicles. Results revealed that a substantial proportion (∼87%) of reads aligned to ribosomal RNA. Of the non-ribosomal RNA sequences, ∼60% aligned to non-coding RNA and repeat sequences including LINE, SINE, satellite repeats, and RNA repeats (tRNA, snRNA, scRNA and srpRNA). The remaining ∼40% of non-ribosomal RNA reads aligned to protein coding genes and splice sites encompassing approximately 13,500 of the known 21,892 protein coding genes of the human genome. Analysis of protein coding genes specific to the renal and genitourinary tract revealed that complete segments of the renal nephron and collecting duct as well as genes indicative of the bladder and prostate could be identified. This study reveals that the entire genitourinary system may be mapped using microvesicle transcript analysis and that the majority of non-ribosomal RNA sequences contained in microvesicles is potentially functional non-coding RNA, which play an emerging role in cell regulation.

Highlights

  • Exosomes and shedding microvesicles are unique forms of vesicles released by all cells into various biofluids [1,2]

  • It has been shown that microvesicles contain nucleic acids including mRNA, miRNA, rRNA and DNA [6,7,8,9], encapsulated from the parent cell cytoplasm during the biogenesis of the microvesicle

  • Such high integrity RNA could be obtained in urine that had been stored for over 5 months at 4uC and 280uC (Russo, unpublished observation) without prior treatment with stabilization buffers indicating the exceptional stability of microvesicles that could not be achieved from urinary cells stored over similar periods of time [8]

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Summary

Introduction

Exosomes (derived from multi-vesicular bodies) and shedding microvesicles (derived from plasma membrane budding) are unique forms of vesicles released by all cells into various biofluids [1,2]. In order to ensure that non-coding RNA species identified using next-generation sequencing techniques are truly RNA derived, DNA digestion of the microvesicle pellet was carried out prior to RNA isolation.

Results
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