Skip to main content

Extracellular RNAs: A New Awareness of Old Perspectives

  • Protocol
Extracellular RNA

Abstract

Extracellular RNA (exRNA) has recently expanded as a highly important area of study in biomarker discovery and cancer therapeutics. exRNA consists of diverse RNA subpopulations that are normally protected from degradation by incorporation into membranous vesicles or by lipid/protein association. They are found circulating in biofluids, and have proven highly promising for minimally invasive diagnostic and prognostic purposes, particularly in oncology. Recent work has made progress in our understanding of exRNAs—from their biogenesis, compartmentalization, and vesicle packaging to their various applications as biomarkers and therapeutics, as well as the new challenges that arise in isolation and purification for accurate and reproducible analysis. Here we review the most recent advancements in exRNA research.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 69.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 89.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 119.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Di Vizio D et al (2012) Large oncosomes in human prostate cancer tissues and in the circulation of mice with metastatic disease. Am J Pathol 181(5):1573–1584

    Article  Google Scholar 

  2. Minciacchi VR, Freeman MR, Di Vizio D (2015) Extracellular vesicles in cancer: exosomes, microvesicles and the emerging role of large oncosomes. Semin Cell Dev Biol 40:41–51

    Article  CAS  Google Scholar 

  3. Yanez-Mo M et al (2015) Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4:27066

    Article  Google Scholar 

  4. Costa-Silva B et al (2015) Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol 17(6):816–826

    Article  CAS  Google Scholar 

  5. Skog J et al (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 10(12):1470–1476

    Article  CAS  Google Scholar 

  6. Chen WW et al (2013) BEAMing and droplet digital PCR analysis of mutant IDH1 mRNA in glioma patient serum and cerebrospinal fluid extracellular vesicles. Mol Ther Nucleic Acids 2:e109

    Article  Google Scholar 

  7. Balaj L et al (2011) Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences. Nat Commun 2:180

    Article  Google Scholar 

  8. Lasser C et al (2011) Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J Transl Med 9:9

    Article  Google Scholar 

  9. Turchinovich A et al (2011) Characterization of extracellular circulating microRNA. Nucleic Acids Res 39(16):7223–7233

    Article  CAS  Google Scholar 

  10. Vickers KC et al (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13(4):423–433

    Article  CAS  Google Scholar 

  11. Crescitelli R et al (2013) Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes. J Extracell Vesicles 2

    Google Scholar 

  12. Lasser C et al (2017) Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing. RNA Biol 14(1):58–72

    Article  Google Scholar 

  13. Bellingham SA, Coleman BM, Hill AF (2012) Small RNA deep sequencing reveals a distinct miRNA signature released in exosomes from prion-infected neuronal cells. Nucleic Acids Res 40(21):10937–10949

    Article  CAS  Google Scholar 

  14. Michell DL et al (2016) Isolation of high-density lipoproteins for non-coding small RNA quantification. J Vis Exp (117)

    Google Scholar 

  15. Arroyo JD et al (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A 108(12):5003–5008

    Article  CAS  Google Scholar 

  16. Cha DJ et al (2015) KRAS-dependent sorting of miRNA to exosomes. Elife 4:e07197

    Article  Google Scholar 

  17. Villarroya-Beltri C et al (2013) Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs. Nat Commun 4:2980

    Article  Google Scholar 

  18. Shurtleff MJ et al (2016) Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction. Elife 5

    Google Scholar 

  19. Yuan T et al (2016) Plasma extracellular RNA profiles in healthy and cancer patients. Sci Rep 6:19413

    Article  CAS  Google Scholar 

  20. Wei Z et al (2016) Fetal bovine serum RNA interferes with the cell culture derived extracellular RNA. Sci Rep 6:31175

    Article  CAS  Google Scholar 

  21. Musilova K, Mraz M (2015) MicroRNAs in B-cell lymphomas: how a complex biology gets more complex. Leukemia 29(5):1004–1017

    Article  CAS  Google Scholar 

  22. Ebert MS, Sharp PA (2012) Roles for microRNAs in conferring robustness to biological processes. Cell 149(3):515–524

    Article  CAS  Google Scholar 

  23. Friedman RC et al (2009) Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 19(1):92–105

    Article  CAS  Google Scholar 

  24. Rodriguez A et al (2004) Identification of mammalian microRNA host genes and transcription units. Genome Res 14(10A):1902–1910

    Article  CAS  Google Scholar 

  25. Pratt AJ, MacRae IJ (2009) The RNA-induced silencing complex: a versatile gene-silencing machine. J Biol Chem 284(27):17897–17901

    Article  CAS  Google Scholar 

  26. Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9(2):102–114

    Article  CAS  Google Scholar 

  27. Eulalio A et al (2009) Deadenylation is a widespread effect of miRNA regulation. RNA 15(1):21–32

    Article  CAS  Google Scholar 

  28. Hutvagner G, Zamore PD (2002) A microRNA in a multiple-turnover RNAi enzyme complex. Science 297(5589):2056–2060

    Article  CAS  Google Scholar 

  29. Nielsen CB et al (2007) Determinants of targeting by endogenous and exogenous microRNAs and siRNAs. RNA 13(11):1894–1910

    Article  CAS  Google Scholar 

  30. Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233

    Article  CAS  Google Scholar 

  31. Chen X et al (2012) Secreted microRNAs: a new form of intercellular communication. Trends Cell Biol 22(3):125–132

    Article  CAS  Google Scholar 

  32. Vickers KC, Remaley AT (2012) Lipid-based carriers of microRNAs and intercellular communication. Curr Opin Lipidol 23(2):91–97

    Article  CAS  Google Scholar 

  33. Melman YF et al (2015) Circulating MicroRNA-30d is associated with response to cardiac resynchronization therapy in heart failure and regulates cardiomyocyte apoptosis: a translational pilot study. Circulation 131(25):2202–2216

    Article  CAS  Google Scholar 

  34. Sharp SJ et al (1985) Structure and transcription of eukaryotic tRNA genes. CRC Crit Rev Biochem 19(2):107–144

    Article  CAS  Google Scholar 

  35. Hurto RL (2011) Unexpected functions of tRNA and tRNA processing enzymes. Adv Exp Med Biol 722:137–155

    Article  CAS  Google Scholar 

  36. Nolte-'t Hoen EN et al (2012) Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions. Nucleic Acids Res 40(18):9272–9285

    Article  CAS  Google Scholar 

  37. Tosar JP et al (2015) Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines. Nucleic Acids Res 43(11):5601–5616

    Article  CAS  Google Scholar 

  38. Baglio SR et al (2015) Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species. Stem Cell Res Ther 6:127

    Article  Google Scholar 

  39. Li M et al (2014) Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers. Philos Trans R Soc Lond Ser B Biol Sci 369(1652)

    Google Scholar 

  40. Vojtech L et al (2014) Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions. Nucleic Acids Res 42(11):7290–7304

    Article  CAS  Google Scholar 

  41. Lunavat TR et al (2015) Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells—evidence of unique microRNA cargos. RNA Biol 12(8):810–823

    Article  Google Scholar 

  42. Lee YS et al (2009) A novel class of small RNAs: tRNA-derived RNA fragments (tRFs). Genes Dev 23(22):2639–2649

    Article  CAS  Google Scholar 

  43. Ivanov P et al (2011) Angiogenin-induced tRNA fragments inhibit translation initiation. Mol Cell 43(4):613–623

    Article  CAS  Google Scholar 

  44. Sobala A, Hutvagner G (2013) Small RNAs derived from the 5′ end of tRNA can inhibit protein translation in human cells. RNA Biol 10(4):553–563

    Article  CAS  Google Scholar 

  45. Freedman JE et al (2016) Diverse human extracellular RNAs are widely detected in human plasma. Nat Commun 7:11106

    Article  CAS  Google Scholar 

  46. Meister G (2013) Argonaute proteins: functional insights and emerging roles. Nat Rev Genet 14(7):447–459

    Article  CAS  Google Scholar 

  47. Hayashi R et al (2016) Genetic and mechanistic diversity of piRNA 3′-end formation. Nature 539(7630):588–592

    Article  CAS  Google Scholar 

  48. Chuma S, Pillai RS (2009) Retrotransposon silencing by piRNAs: ping-pong players mark their sub-cellular boundaries. PLoS Genet 5(12):e1000770

    Article  Google Scholar 

  49. Kowalski MP, Krude T (2015) Functional roles of non-coding Y RNAs. Int J Biochem Cell Biol 66:20–29

    Article  CAS  Google Scholar 

  50. Chen X et al (2013) An RNA degradation machine sculpted by Ro autoantigen and noncoding RNA. Cell 153(1):166–177

    Article  CAS  Google Scholar 

  51. Krude T et al (2009) Y RNA functions at the initiation step of mammalian chromosomal DNA replication. J Cell Sci 122(Pt 16):2836–2845

    Article  CAS  Google Scholar 

  52. Nicolas FE et al (2012) Biogenesis of Y RNA-derived small RNAs is independent of the microRNA pathway. FEBS Lett 586(8):1226–1230

    Article  CAS  Google Scholar 

  53. Meiri E et al (2010) Discovery of microRNAs and other small RNAs in solid tumors. Nucleic Acids Res 38(18):6234–6246

    Article  CAS  Google Scholar 

  54. Chakrabortty SK et al (2015) Extracellular vesicle-mediated transfer of processed and functional RNY5 RNA. RNA 21(11):1966–1979

    Article  CAS  Google Scholar 

  55. Kohn M et al (2015) The Y3** ncRNA promotes the 3′ end processing of histone mRNAs. Genes Dev 29(19):1998–2003

    Article  Google Scholar 

  56. Wei Z, Batagov AO, Schinelli S, Wang J, Wang Y, El Fatimy R, Rabinovsky R, Balaj L, Chen CC, Hochberg F, Carter B, Breakefield XO, Krichevsky AM (2017) Coding and noncoding landscape of extracellular RNA released by human glioma stem cells. Nat Commun 8(1):1145

    Google Scholar 

  57. Yeri A et al (2017) Total extracellular small RNA profiles from plasma, saliva, and urine of healthy subjects. Sci Rep 7:44061

    Article  CAS  Google Scholar 

  58. Huang X et al (2013) Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC Genomics 14:319

    Article  CAS  Google Scholar 

  59. Hansen TB et al (2013) Natural RNA circles function as efficient microRNA sponges. Nature 495(7441):384–388

    Article  CAS  Google Scholar 

  60. Morris KV, Mattick JS (2014) The rise of regulatory RNA. Nat Rev Genet 15(6):423–437

    Article  CAS  Google Scholar 

  61. Smith MA et al (2013) Widespread purifying selection on RNA structure in mammals. Nucleic Acids Res 41(17):8220–8236

    Article  CAS  Google Scholar 

  62. Johnsson P et al (2014) Evolutionary conservation of long non-coding RNAs; sequence, structure, function. Biochim Biophys Acta 1840(3):1063–1071

    Article  CAS  Google Scholar 

  63. Kogure T et al (2013) Extracellular vesicle-mediated transfer of a novel long noncoding RNA TUC339: a mechanism of intercellular signaling in human hepatocellular cancer. Genes Cancer 4(7–8):261–272

    Article  CAS  Google Scholar 

  64. Maxwell ES, Fournier MJ (1995) The small nucleolar RNAs. Annu Rev Biochem 64:897–934

    Article  CAS  Google Scholar 

  65. Appaiah HN et al (2011) Persistent upregulation of U6:SNORD44 small RNA ratio in the serum of breast cancer patients. Breast Cancer Res 13(5):R86

    Article  CAS  Google Scholar 

  66. Chen LL (2016) The biogenesis and emerging roles of circular RNAs. Nat Rev Mol Cell Biol 17(4):205–211

    Article  CAS  Google Scholar 

  67. Memczak S et al (2013) Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495(7441):333–338

    Article  CAS  Google Scholar 

  68. Turchinovich A, Burwinkel B (2012) Distinct AGO1 and AGO2 associated miRNA profiles in human cells and blood plasma. RNA Biol 9(8):1066–1075

    Article  CAS  Google Scholar 

  69. Shelke GV et al (2014) Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum. J Extracell Vesicles 3

    Google Scholar 

  70. Mitchell PS et al (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 105(30):10513–10518

    Article  CAS  Google Scholar 

  71. Brunet-Vega A et al (2015) Variability in microRNA recovery from plasma: comparison of five commercial kits. Anal Biochem 488:28–35

    Article  CAS  Google Scholar 

  72. Li X, Mauro M, Williams Z (2015) Comparison of plasma extracellular RNA isolation kits reveals kit-dependent biases. Biotechniques 59(1):13–17

    Article  CAS  Google Scholar 

  73. Royo F et al (2016) Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples. J Extracell Vesicles 5:29497

    Article  Google Scholar 

  74. Van Deun J et al (2014) The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J Extracell Vesicles 3

    Google Scholar 

  75. Laurent LC et al (2015) Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium. J Extracell Vesicles 4:26533

    Article  Google Scholar 

  76. Tanriverdi K et al (2016) Comparison of RNA isolation and associated methods for extracellular RNA detection by high-throughput quantitative polymerase chain reaction. Anal Biochem 501:66–74

    Article  CAS  Google Scholar 

  77. Gardiner C et al (2016) Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey. J Extracell Vesicles 5:32945

    Article  Google Scholar 

  78. Jeppesen DK et al (2014) Comparative analysis of discrete exosome fractions obtained by differential centrifugation. J Extracell Vesicles 3:25011

    Article  Google Scholar 

  79. Yuana Y et al (2014) Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation. J Extracell Vesicles 3

    Google Scholar 

  80. Boing AN et al (2014) Single-step isolation of extracellular vesicles by size-exclusion chromatography. J Extracell Vesicles 3

    Google Scholar 

  81. Vergauwen G et al (2017) Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. Sci Rep 7(1):2704

    Article  Google Scholar 

  82. Gamez-Valero A et al (2016) Size-exclusion chromatography-based isolation minimally alters extracellular vesicles’ characteristics compared to precipitating agents. Sci Rep 6:33641

    Article  CAS  Google Scholar 

  83. Kanwar SS et al (2014) Microfluidic device (ExoChip) for on-chip isolation, quantification and characterization of circulating exosomes. Lab Chip 14(11):1891–1900

    Article  CAS  Google Scholar 

  84. Balaj L et al (2015) Heparin affinity purification of extracellular vesicles. Sci Rep 5:10266

    Article  CAS  Google Scholar 

  85. Enderle D et al (2015) Characterization of RNA from exosomes and other extracellular vesicles isolated by a novel spin column-based method. PLoS One 10(8):e0136133

    Article  Google Scholar 

  86. Gallart-Palau X et al (2015) Extracellular vesicles are rapidly purified from human plasma by PRotein Organic Solvent PRecipitation (PROSPR). Sci Rep 5:14664

    Article  CAS  Google Scholar 

  87. Shih CL et al (2016) Development of a magnetic bead-based method for the collection of circulating extracellular vesicles. New Biotechnol 33(1):116–122

    Article  CAS  Google Scholar 

  88. Puhka M et al (2017) KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine. Eur J Pharm Sci 98:30–39

    Article  CAS  Google Scholar 

  89. Turchinovich A, Weiz L, Burwinkel B (2013) Isolation of circulating microRNA associated with RNA-binding protein. Methods Mol Biol 1024:97–107

    Article  CAS  Google Scholar 

  90. Thery C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9(8):581–593

    Article  CAS  Google Scholar 

  91. Schipper HM et al (2007) MicroRNA expression in Alzheimer blood mononuclear cells. Gene Regul Syst Bio 1:263–274

    PubMed  PubMed Central  Google Scholar 

  92. Karlsson O et al (2016) Detection of long non-coding RNAs in human breastmilk extracellular vesicles: implications for early child development. Epigenetics:0

    Google Scholar 

  93. Machtinger R et al (2017) Extracellular microRNAs in follicular fluid and their potential association with oocyte fertilization and embryo quality: an exploratory study. J Assist Reprod Genet 34(4):525–533

    Article  Google Scholar 

  94. Dear JW, Street JM, Bailey MA (2013) Urinary exosomes: a reservoir for biomarker discovery and potential mediators of intrarenal signalling. Proteomics 13(10–11):1572–1580

    Article  CAS  Google Scholar 

  95. Quinn JF et al (2015) Extracellular RNAs: development as biomarkers of human disease. J Extracell Vesicles 4:27495

    Article  Google Scholar 

  96. March-Villalba JA et al (2012) Cell-free circulating plasma hTERT mRNA is a useful marker for prostate cancer diagnosis and is associated with poor prognosis tumor characteristics. PLoS One 7(8):e43470

    Article  CAS  Google Scholar 

  97. Xie Z et al (2013) Salivary microRNAs as promising biomarkers for detection of esophageal cancer. PLoS One 8(4):e57502

    Article  CAS  Google Scholar 

  98. Xie Z et al (2015) Salivary microRNAs show potential as a noninvasive biomarker for detecting resectable pancreatic cancer. Cancer Prev Res (Phila) 8(2):165–173

    Article  CAS  Google Scholar 

  99. Akers JC et al (2013) MiR-21 in the extracellular vesicles (EVs) of cerebrospinal fluid (CSF): a platform for glioblastoma biomarker development. PLoS One 8(10):e78115

    Article  CAS  Google Scholar 

  100. Akat KM et al (2014) Comparative RNA-sequencing analysis of myocardial and circulating small RNAs in human heart failure and their utility as biomarkers. Proc Natl Acad Sci U S A 111(30):11151–11156

    Article  CAS  Google Scholar 

  101. de Gonzalo-Calvo D et al (2016) Circulating long-non coding RNAs as biomarkers of left ventricular diastolic function and remodelling in patients with well-controlled type 2 diabetes. Sci Rep 6:37354

    Article  Google Scholar 

  102. Ben-Dov IZ et al (2016) Cell and microvesicle urine microRNA deep sequencing profiles from healthy individuals: observations with potential impact on biomarker studies. PLoS One 11(1):e0147249

    Article  Google Scholar 

  103. Rodosthenous RS, Burris HH, Sanders AP, Just AC, Dereix AE, Svensson K, Solano M, Téllez-Rojo MM, Wright RO, Baccarelli AA (2017) Second trimester extracellular microRNAs in maternal blood and fetal growth: an exploratory study. Epigenetics 12(9):804–810

    Google Scholar 

  104. Tsochandaridis M, Nasca L, Toga C, Levy-Mozziconacci A (2015) Circulating MicroRNAs as clinical biomarkers in the predictions of pregnancy complications. BioMed Res Int 2015:294954

    Google Scholar 

  105. Ohno S et al (2013) Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol Ther 21(1):185–191

    Article  CAS  Google Scholar 

  106. Zhang Y et al (2014) Microvesicle-mediated delivery of transforming growth factor beta1 siRNA for the suppression of tumor growth in mice. Biomaterials 35(14):4390–4400

    Article  CAS  Google Scholar 

  107. Jinek M, Doudna JA (2009) A three-dimensional view of the molecular machinery of RNA interference. Nature 457(7228):405–412

    Article  CAS  Google Scholar 

  108. Lai RC, Yeo RW, Lim SK (2015) Mesenchymal stem cell exosomes. Semin Cell Dev Biol 40:82–88

    Article  CAS  Google Scholar 

  109. Shimbo K et al (2014) Exosome-formed synthetic microRNA-143 is transferred to osteosarcoma cells and inhibits their migration. Biochem Biophys Res Commun 445(2):381–387

    Article  CAS  Google Scholar 

  110. Lou G et al (2015) Exosomes derived from miR-122-modified adipose tissue-derived MSCs increase chemosensitivity of hepatocellular carcinoma. J Hematol Oncol 8:122

    Article  Google Scholar 

  111. Greco KA et al (2016) PLK-1 silencing in bladder cancer by siRNA delivered with exosomes. Urology 91:241.e1–241.e7

    Article  Google Scholar 

  112. Mizrak A et al (2013) Genetically engineered microvesicles carrying suicide mRNA/protein inhibit schwannoma tumor growth. Mol Ther 21(1):101–108

    Article  CAS  Google Scholar 

Download references

Acknowledgements

B.G. is an Edward R. and Anne G. Lefler Center Postdoctoral Fellow. The authors declare no conflicts of interest.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Leonora Balaj .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Sadik, N. et al. (2018). Extracellular RNAs: A New Awareness of Old Perspectives. In: Patel, T. (eds) Extracellular RNA. Methods in Molecular Biology, vol 1740. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7652-2_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7652-2_1

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7651-5

  • Online ISBN: 978-1-4939-7652-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics