REFERENCES
1. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993;75:843-54.
2. Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell. 1993;75:855-62.
3. Pasquinelli AE, Reinhart BJ, Slack F, et al. Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA. Nature. 2000;408:86-9.
4. Reinhart BJ, Slack FJ, Basson M, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature. 2000;403:901-6.
6. Grimson A, Srivastava M, Fahey B, et al. Early origins and evolution of microRNAs and Piwi-interacting RNAs in animals. Nature. 2008;455:1193-7.
7. Zheng Y, Cai X, Bradley JE. microRNAs in parasites and parasite infection. RNA Biol. 2013;10:371-9.
8. Nobel Prize in Physiology or Medicine 2024. Available from: https://www.nobelprize.org/prizes/medicine/2024/summary/. [Last accessed on 28 May 2026].
10. Bofill-De Ros X, Vang Ørom UA. Recent progress in miRNA biogenesis and decay. RNA Biol. 2024;21:1-8.
11. Daugaard I, Hansen TB. Biogenesis and function of Ago-associated RNAs. Trends Genet. 2017;33:208-19.
12. Salim U, Kumar A, Kulshreshtha R, Vivekanandan P. Biogenesis, characterization, and functions of mirtrons. Wiley Interdiscip Rev RNA. 2022;13:e1680.
13. Khanal S, de Cruz M, Strickland B, Mansfield K, Lai EC, Flynt A. A tailed mirtron promotes longevity in Drosophila. Nucleic Acids Res. 2024;52:1080-9.
14. Ruby JG, Jan CH, Bartel DP. Intronic microRNA precursors that bypass Drosha processing. Nature. 2007;448:83-6.
15. Muthukumar S, Li CT, Liu RJ, Bellodi C. Roles and regulation of tRNA-derived small RNAs in animals. Nat Rev Mol Cell Biol. 2024;25:359-78.
17. Liu M, Cho WC, Flynn RJ, Jin X, Song H, Zheng Y. microRNAs in parasite-induced liver fibrosis: from mechanisms to diagnostics and therapeutics. Trends Parasitol. 2023;39:859-72.
18. Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol. 2011;13:423-33.
19. Liebana-Jordan M, Brotons B, Falcon-Perez JM, Gonzalez E. Extracellular vesicles in the fungi kingdom. Int J Mol Sci. 2021;22:7221.
20. Maricchiolo E, Creanza P, Osnato M, et al. Plant vs mammal extracellular vesicles: new tools in therapeutic drug delivery. Curr Res Biotechnol. 2026;11:100352.
21. Sartorio MG, Pardue EJ, Feldman MF, Haurat MF. Bacterial outer membrane vesicles: from discovery to applications. Annu Rev Microbiol. 2021;75:609-30.
22. Wang X, Thompson CD, Weidenmaier C, Lee JC. Release of Staphylococcus aureus extracellular vesicles and their application as a vaccine platform. Nat Commun. 2018;9:1379.
23. Jeppesen DK, Fenix AM, Franklin JL, et al. Reassessment of exosome composition. Cell. 2019;177:428-45.e18.
24. Xu D, Di K, Fan B, et al. MicroRNAs in extracellular vesicles: sorting mechanisms, diagnostic value, isolation, and detection technology. Front Bioeng Biotechnol. 2022;10:948959.
25. Zhong Y, Wang X, Zhao X, et al. Multifunctional milk-derived small extracellular vesicles and their biomedical applications. Pharmaceutics. 2023;15:1418.
26. Berumen Sánchez G, Bunn KE, Pua HH, Rafat M. Extracellular vesicles: mediators of intercellular communication in tissue injury and disease. Cell Commun Signal. 2021;19:104.
28. Yu J, Sane S, Kim JE, et al. Biogenesis and delivery of extracellular vesicles: harnessing the power of EVs for diagnostics and therapeutics. Front Mol Biosci. 2023;10:1330400.
29. Sun M, Xue X, Li L, et al. Ectosome biogenesis and release processes observed by using live-cell dynamic imaging in mammalian glial cells. Quant Imaging Med Surg. 2021;11:4604-16.
30. Dixson AC, Dawson TR, Di Vizio D, Weaver AM. Context-specific regulation of extracellular vesicle biogenesis and cargo selection. Nat Rev Mol Cell Biol. 2023;24:454-76.
31. Gurunathan S, Kang MH, Qasim M, Khan K, Kim JH. Biogenesis, membrane trafficking, functions, and next generation nanotherapeutics medicine of extracellular vesicles. Int J Nanomedicine. 2021;16:3357-83.
32. van Niel G, D’Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19:213-28.
33. Juan T, Fürthauer M. Biogenesis and function of ESCRT-dependent extracellular vesicles. Semin Cell Dev Biol. 2018;74:66-77.
34. Bebelman MP, Janssen E, Pegtel DM, Crudden C. The forces driving cancer extracellular vesicle secretion. Neoplasia. 2021;23:149-57.
35. Kalra H, Drummen GP, Mathivanan S. Focus on extracellular vesicles: introducing the next small big thing. Int J Mol Sci. 2016;17:170.
36. Phan TK, Fonseka P, Tixeira R, et al. Pannexin-1 channel regulates nuclear content packaging into apoptotic bodies and their size. Proteomics. 2021;21:e2000097.
37. Shi B, Tang C, Rutter SF, et al. NINJ1 oligomerises on large apoptotic cell-derived extracellular vesicles to regulate vesicle stability and cellular content release. Front Immunol. 2025;16:1599809.
38. Kakarla R, Hur J, Kim YJ, Kim J, Chwae YJ. Apoptotic cell-derived exosomes: messages from dying cells. Exp Mol Med. 2020;52:1-6.
39. Chen TY, Gonzalez-Kozlova E, Soleymani T, et al. Extracellular vesicles carry distinct proteo-transcriptomic signatures that are different from their cancer cell of origin. iScience. 2022;25:104414.
40. Dogra N, Chen TY, Gonzalez-Kozlova E, et al. Extracellular vesicles carry transcriptional ‘dark matter’ revealing tissue-specific information. J Extracell Vesicles. 2024;13:e12481.
41. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9:654-9.
42. Cheng L, Vella LJ, Barnham KJ, McLean C, Masters CL, Hill AF. Small RNA fingerprinting of Alzheimer’s disease frontal cortex extracellular vesicles and their comparison with peripheral extracellular vesicles. J Extracell Vesicles. 2020;9:1766822.
43. Liu XM, Ma L, Schekman R. Selective sorting of microRNAs into exosomes by phase-separated YBX1 condensates. Elife. 2021;10:E71982.
44. Miceli RT, Chen TY, Nose Y, et al. Extracellular vesicles, RNA sequencing, and bioinformatic analyses: challenges, solutions, and recommendations. J Extracell Vesicles. 2024;13:e70005.
45. Murillo OD, Thistlethwaite W, Rozowsky J, et al. exRNA atlas analysis reveals distinct extracellular RNA cargo types and their carriers present across human biofluids. Cell. 2019;177:463-77.e15.
46. Nolte-'t Hoen EN, Buermans HP, Waasdorp M, Stoorvogel W, Wauben MH, 't Hoen PA. 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. 2012;40:9272-85.
47. Zubor P, Kubatka P, Kajo K, et al. Why the gold standard approach by mammography demands extension by multiomics? Application of liquid biopsy miRNA profiles to breast cancer disease management. Int J Mol Sci. 2019;20:2878.
48. Poddar S, Brozzi F, Cosentino C, et al. Role of small intronic RNAs in the crosstalk between immune cells and β-cells during type 1 diabetes development. RNA Biol. 2026;23:1-15.
49. Tkach M, Thalmensi J, Timperi E, et al. Extracellular vesicles from triple negative breast cancer promote pro-inflammatory macrophages associated with better clinical outcome. Proc Natl Acad Sci U S A. 2022;119:e2107394119.
50. Abdelgawad A, Huang Y, Gololobova O, et al. Defining the parameters for sorting of RNA cargo into extracellular vesicles. J Extracell Vesicles. 2025;14:e70113.
51. Garcia-Martin R, Wang G, Brandão BB, et al. MicroRNA sequence codes for small extracellular vesicle release and cellular retention. Nature. 2022;601:446-51.
52. Koppers-Lalic D, Hackenberg M, Bijnsdorp IV, et al. Nontemplated nucleotide additions distinguish the small RNA composition in cells from exosomes. Cell Rep. 2014;8:1649-58.
53. Shurtleff MJ, Temoche-Diaz MM, Karfilis KV, Ri S, Schekman R. Y-box protein 1 is required to sort microRNAs into exosomes in cells and in a cell-free reaction. Elife. 2016;5:e19276.
54. Villarroya-Beltri C, Gutiérrez-Vázquez C, Sánchez-Cabo F, et al. Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs. Nat Commun. 2013;4:2980.
55. Martellucci S, Orefice NS, Angelucci A, Luce A, Caraglia M, Zappavigna S. Extracellular vesicles: new endogenous shuttles for miRNAs in cancer diagnosis and therapy? Int J Mol Sci. 2020;21:6486.
56. Bellingham SA, Coleman BM, Hill AF. Small RNA deep sequencing reveals a distinct miRNA signature released in exosomes from prion-infected neuronal cells. Nucleic Acids Res. 2012;40:10937-49.
57. Admyre C, Johansson SM, Qazi KR, et al. Exosomes with immune modulatory features are present in human breast milk. J Immunol. 2007;179:1969-78.
58. Atukorala I, Hannan N, Hui L. Immersed in a reservoir of potential: amniotic fluid-derived extracellular vesicles. J Transl Med. 2024;22:348.
59. Bartel S, Wolters JC, Noor H, et al. Altered extracellular vesicle-derived protein and microRNA signatures in bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease. Cells. 2024;13:945.
60. Davidson KR, Ha DM, Schwarz MI, Chan ED. Bronchoalveolar lavage as a diagnostic procedure: a review of known cellular and molecular findings in various lung diseases. J Thorac Dis. 2020;12:4991-5019.
61. Golan-Gerstl R, Elbaum Shiff Y, Moshayoff V, Schecter D, Leshkowitz D, Reif S. Characterization and biological function of milk-derived miRNAs. Mol Nutr Food Res. 2017;61:1700009.
62. Grigor’eva AE, Tamkovich SN, Eremina AV, et al. Exosomes in tears of healthy individuals: isolation, identification, and characterization. Biochem Moscow Suppl Ser B. 2016;10:165-72.
63. Johnson J, Shojaee M, Mitchell Crow J, Khanabdali R. From mesenchymal stromal cells to engineered extracellular vesicles: a new therapeutic paradigm. Front Cell Dev Biol. 2021;9:705676.
64. Keller S, Ridinger J, Rupp AK, Janssen JW, Altevogt P. Body fluid derived exosomes as a novel template for clinical diagnostics. J Transl Med. 2011;9:86.
65. Moelans CB, Patuleia SIS, van Gils CH, van der Wall E, van Diest PJ. Application of nipple aspirate fluid miRNA profiles for early breast cancer detection and management. Int J Mol Sci. 2019;20:5814.
66. Mussack V, Pfaffl MW. Impact of autologous blood transfusions on surface marker and microRNA profiles of urinary extracellular vesicles. Extracell Vesicles Circ Nucl Acids. 2025;6:876-94.
67. Mussack V, Wittmann G, Pfaffl MW. Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing-Enabling robust and non-invasive biomarker research. Biomol Detect Quantif. 2019;17:100089.
69. Papineni RS, Rosenthal FS. The size distribution of droplets in the exhaled breath of healthy human subjects. J Aerosol Med. 1997;10:105-16.
70. Simpson MR, Brede G, Johansen J, et al. Human breast milk miRNA, maternal probiotic supplementation and atopic dermatitis in offspring. PLoS One. 2015;10:e0143496.
71. Tietje A, Maron KN, Wei Y, Feliciano DM. Cerebrospinal fluid extracellular vesicles undergo age dependent declines and contain known and novel non-coding RNAs. PLoS One. 2014;9:e113116.
72. Zhou H, Yuen PS, Pisitkun T, et al. Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery. Kidney Int. 2006;69:1471-6.
73. Bettio V, Mazzucco E, Antona A, et al. Extracellular vesicles from human plasma for biomarkers discovery: impact of anticoagulants and isolation techniques. PLoS One. 2023;18:e0285440.
74. Dilsiz N. A comprehensive review on recent advances in exosome isolation and characterization: toward clinical applications. Transl Oncol. 2024;50:102121.
75. Reithmair M, Lindemann A, Mussack V, Pfaffl MW. Isolation and characterization of urinary extracellular vesicles for microRNA biomarker signature development with reference to MISEV compliance. Methods Mol Biol. 2022;2504:113-33.
76. Simon NK, Volz S, Rios dlRRJ, Wedig T, Montigel SH, et al. Preanalytical framework for routine clinical use of liquid biopsies: combining EVs and cfDNA. Extracell Vesicles Circ Nucl Acids. 2025;6:626-50.
77. Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006;Chapter 3:Unit 3.22.
78. Welsh JA, Goberdhan DCI, O’Driscoll L, et al.; MISEV Consortium. Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches. J Extracell Vesicles. 2024;13:e12404.
79. Zhang G, Ding Y, Zhang H, et al. Assessment of urine sample collection and processing variables for extracellular vesicle-based proteomics. Analyst. 2024;149:3416-24.
80. Köberle V, Kakoschky B, Ibrahim AA, et al. Vesicle-associated microRNAs are released from blood cells on incubation of blood samples. Transl Res. 2016;169:40-6.
81. Ma C, Ding R, Hao K, et al. Storage stability of blood samples for miRNAs in glycosylated extracellular vesicles. Molecules. 2023;29:103.
82. Kim HJ, Rames MJ, Tassi Yunga S, et al. Irreversible alteration of extracellular vesicle and cell-free messenger RNA profiles in human plasma associated with blood processing and storage. Sci Rep. 2022;12:2099.
83. Barreiro K, Dwivedi OP, Valkonen S, et al. Urinary extracellular vesicles: assessment of pre-analytical variables and development of a quality control with focus on transcriptomic biomarker research. J Extracell Vesicles. 2021;10:e12158.
84. Lee J, Kim E, Park J, Choi S, Lee MS, Park J. Pre-analytical handling conditions and protein marker recovery from urine extracellular vesicles for bladder cancer diagnosis. PLoS One. 2023;18:e0291198.
85. Licatini LM, Licatini LM, Haddadin FA, et al. Pre-analytical characterization of CNS-derived extracellular vesicles from human saliva: effect of room temperature and cellular origin. Front Neurosci. 2026;20:1765229.
86. Boulestreau J, Molina L, Ouedraogo A, et al. Salivary extracellular vesicles isolation methods impact the robustness of downstream biomarkers detection. Sci Rep. 2024;14:31233.
87. Cho YE, Vorn R, Chimenti M, et al. Extracellular vesicle miRNAs in breast milk of obese mothers. Front Nutr. 2022;9:976886.
88. Ma L, Huo Y, Tang Q, et al. Human breast milk exosomal miRNAs are influenced by premature delivery and affect neurodevelopment. Mol Nutr Food Res. 2024;68:e2300113.
89. van Herwijnen MJ, Zonneveld MI, Goerdayal S, et al. Comprehensive proteomic analysis of human milk-derived extracellular vesicles unveils a novel functional proteome distinct from other milk components. Mol Cell Proteomics. 2016;15:3412-23.
91. Chung AC, Huang XR, Meng X, Lan HY. miR-192 mediates TGF-beta/Smad3-driven renal fibrosis. J Am Soc Nephrol. 2010;21:1317-25.
93. Franczyk B, Gluba-Brzózka A, Olszewski R, Parolczyk M, Rysz-Górzyńska M, Rysz J. miRNA biomarkers in renal disease. Int Urol Nephrol. 2022;54:575-88.
94. Grätz C, Schuster M, Brandes F, et al. A pipeline for the development and analysis of extracellular vesicle-based transcriptomic biomarkers in molecular diagnostics. Mol Aspects Med. 2024;97:101269.
95. Inubushi S, Kawaguchi H, Mizumoto S, et al. Oncogenic miRNAs identified in tear exosomes from metastatic breast cancer patients. Anticancer Res. 2020;40:3091-6.
96. Karvinen S, Sievänen T, Karppinen JE, et al. MicroRNAs in extracellular vesicles in sweat change in response to endurance exercise. Front Physiol. 2020;11:676.
97. Lin J, Huang X, Zhang J, et al. Amniotic fluid-derived exosomal miR-146a-5p ameliorates preeclampsia phenotypes by inhibiting HIF-1α/FLT-1 expression. Placenta. 2025;162:35-44.
98. Lu Y, Liu D, Feng Q, Liu Z. Diabetic nephropathy: perspective on extracellular vesicles. Front Immunol. 2020;11:943.
99. Lv W, Fan F, Wang Y, et al. Therapeutic potential of microRNAs for the treatment of renal fibrosis and CKD. Physiol Genomics. 2018;50:20-34.
100. Nonaka T, Wong DTW. Saliva-exosomics in cancer: molecular characterization of cancer-derived exosomes in saliva. Enzymes. 2017;42:125-51.
101. Wu J, Liu G, Jia R, Guo J. Salivary extracellular vesicles: biomarkers and beyond in human diseases. Int J Mol Sci. 2023;24:17328.
102. Xie Y, Jia Y, Cuihua X, Hu F, Xue M, Xue Y. Urinary exosomal microRNA profiling in incipient type 2 diabetic kidney disease. J Diabetes Res. 2017;2017:6978984.
103. Ali N, Rahat ST, Mäkelä M, et al. Metabolic patterns of sweat-extracellular vesicles during exercise and recovery states using clinical grade patches. Front Physiol. 2023;14:1295852.
104. An J, McDowell A, Kim YK, Kim TB. Extracellular vesicle-derived microbiome obtained from exhaled breath condensate in patients with asthma. Ann Allergy Asthma Immunol. 2021;126:729-31.
105. Bano A, Yadav P, Sharma M, et al. Extraction and characterization of exosomes from the exhaled breath condensate and sputum of lung cancer patients and vulnerable tobacco consumers-potential noninvasive diagnostic biomarker source. J Breath Res. 2024;18:046003.
106. Batochir C, Kim IA, Jo EJ, et al. Discrimination of lung cancer and benign lung diseases using BALF exosome DNA methylation profile. Cancers. 2024;16:2765.
107. Dobhal G, Datta A, Ayupova D, Teesdale-Spittle P, Goreham RV. Isolation, characterisation and detection of breath-derived extracellular vesicles. Sci Rep. 2020;10:17381.
108. Ghosh N, Choudhury P, Joshi M, et al. Global metabolome profiling of exhaled breath condensates in male smokers with asthma COPD overlap and prediction of the disease. Sci Rep. 2021;11:16664.
109. Han W, Wang T, Reilly AA, Keller SM, Spivack SD. Gene promoter methylation assayed in exhaled breath, with differences in smokers and lung cancer patients. Respir Res. 2009;10:86.
110. He J, Ao C, Li M, et al. Clusterin-carrying extracellular vesicles derived from human umbilical cord mesenchymal stem cells restore the ovarian function of premature ovarian failure mice through activating the PI3K/AKT pathway. Stem Cell Res Ther. 2024;15:300.
111. Kim JE, Eom JS, Kim WY, et al. Diagnostic value of microRNAs derived from exosomes in bronchoalveolar lavage fluid of early-stage lung adenocarcinoma: a pilot study. Thorac Cancer. 2018;9:911-5.
112. Kubáň P, Foret F. Exhaled breath condensate: determination of non-volatile compounds and their potential for clinical diagnosis and monitoring. a review. Anal Chim Acta. 2013;805:1-18.
113. Lacombe M, Marie-Desvergne C, Combes F, et al. Proteomic characterization of human exhaled breath condensate. J Breath Res. 2018;12:021001.
114. Lee SE, Park HY, Hur JY, et al. Genomic profiling of extracellular vesicle-derived DNA from bronchoalveolar lavage fluid of patients with lung adenocarcinoma. Transl Lung Cancer Res. 2021;10:104-16.
115. Lucchetti D, Santini G, Perelli L, et al. Detection and characterisation of extracellular vesicles in exhaled breath condensate and sputum of COPD and severe asthma patients. Eur Respir J. 2021;58:2003024.
116. Mazzone PJ. Analysis of volatile organic compounds in the exhaled breath for the diagnosis of lung cancer. J Thorac Oncol. 2008;3:774-80.
117. Mitchell MI, Ben-Dov IZ, Ye K, et al. Exhaled breath condensate contains extracellular vesicles (EVs) that carry miRNA cargos of lung tissue origin that can be selectively purified and analyzed. J Extracell Vesicles. 2024;13:e12440.
118. Moura PC, Raposo M, Vassilenko V. Breath volatile organic compounds (VOCs) as biomarkers for the diagnosis of pathological conditions: a review. Biomed J. 2023;46:100623.
119. Ratiu IA, Ligor T, Bocos-Bintintan V, Mayhew CA, Buszewski B. Volatile organic compounds in exhaled breath as fingerprints of lung cancer, asthma and COPD. J Clin Med. 2020;10:32.
120. Sanak M, Gielicz A, Bochenek G, Kaszuba M, Niżankowska-Mogilnicka E, Szczeklik A. Targeted eicosanoid lipidomics of exhaled breath condensate provide a distinct pattern in the aspirin-intolerant asthma phenotype. J Allergy Clin Immunol. 2011;127:1141-7.e2.
121. Sinha A, Yadav AK, Chakraborty S, et al. Exosome-enclosed microRNAs in exhaled breath hold potential for biomarker discovery in patients with pulmonary diseases. J Allergy Clin Immunol. 2013;132:219-22.
122. Sun C, Zhou T, Xie G, et al. Proteomics of exhaled breath condensate in stable COPD and non-COPD controls using tandem mass tags (TMTs) quantitative mass spectrometry: a pilot study. J Proteomics. 2019;206:103392.
123. Xiao P, Chen JR, Zhou F, et al. Methylation of P16 in exhaled breath condensate for diagnosis of non-small cell lung cancer. Lung Cancer. 2014;83:56-60.
124. Yang Ai SS, Hsu K, Herbert C, et al. Mitochondrial DNA mutations in exhaled breath condensate of patients with lung cancer. Respir Med. 2013;107:911-8.
125. Youssef O, Knuuttila A, Piirilä P, Böhling T, Sarhadi V, Knuutila S. Presence of cancer-associated mutations in exhaled breath condensates of healthy individuals by next generation sequencing. Oncotarget. 2017;8:18166-76.
126. Zareba L, Szymanski J, Homoncik Z, Czystowska-Kuzmicz M. EVs from BALF-mediators of inflammation and potential biomarkers in lung diseases. Int J Mol Sci. 2021;22:3651.
127. Faversani A, Favero C, Dioni L, et al. An EBC/plasma miRNA signature discriminates lung adenocarcinomas from pleural mesothelioma and healthy controls. Front Oncol. 2021;11:643280.
128. Pinkerton M, Chinchilli V, Banta E, et al. Differential expression of microRNAs in exhaled breath condensates of patients with asthma, patients with chronic obstructive pulmonary disease, and healthy adults. J Allergy Clin Immunol. 2013;132:217-9.
129. Qin B, Hu XM, Su ZH, Zeng XB, Ma HY, Xiong K. Tissue-derived extracellular vesicles: research progress from isolation to application. Pathol Res Pract. 2021;226:153604.
130. Lee JC, Ray RM, Scott TA. Prospects and challenges of tissue-derived extracellular vesicles. Mol Ther. 2024;32:2950-78.
131. Martins TS, Vaz M, Henriques AG. A review on comparative studies addressing exosome isolation methods from body fluids. Anal Bioanal Chem. 2023;415:1239-63.
132. Zhou B, Xu K, Zheng X, et al. Application of exosomes as liquid biopsy in clinical diagnosis. Signal Transduct Target Ther. 2020;5:144.
134. Sands J, Tammemägi MC, Couraud S, et al. Lung screening benefits and challenges: a review of the data and outline for implementation. J Thorac Oncol. 2021;16:37-53.
135. Dama E, Colangelo T, Fina E, et al. Biomarkers and lung cancer early detection: state of the art. Cancers. 2021;13:3919.
136. Seijo LM, Peled N, Ajona D, et al. Biomarkers in lung cancer screening: achievements, promises, and challenges. J Thorac Oncol. 2019;14:343-57.
137. Billatos E, Vick JL, Lenburg ME, Spira AE. The airway transcriptome as a biomarker for early lung cancer detection. Clin Cancer Res. 2018;24:2984-92.
138. Bahmer T, Krauss-Etschmann S, Buschmann D, et al. RNA-seq-based profiling of extracellular vesicles in plasma reveals a potential role of miR-122-5p in asthma. Allergy. 2021;76:366-71.
139. Rufo J, Zhang P, Wang Z, et al. High-yield and rapid isolation of extracellular vesicles by flocculation via orbital acoustic trapping: FLOAT. Microsyst Nanoeng. 2024;10:23.
140. Patel PH, Antoine MH, Sankari A, Ullah S. Bronchoalveolar lavage. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2026. Available from: https://www.ncbi.nlm.nih.gov/books/NBK430762/. [Last accessed on 28 May 2026].
141. Carnino JM, Lee H, Jin Y. Isolation and characterization of extracellular vesicles from Broncho-alveolar lavage fluid: a review and comparison of different methods. Respir Res. 2019;20:240.
142. Mohan A, Agarwal S, Clauss M, Britt NS, Dhillon NK. Extracellular vesicles: novel communicators in lung diseases. Respir Res. 2020;21:175.
143. Khoubnasabjafari M, Mogaddam MRA, Rahimpour E, Soleymani J, Saei AA, Jouyban A. Breathomics: review of sample collection and analysis, data modeling and clinical applications. Crit Rev Anal Chem. 2022;52:1461-87.
144. Lawal O, Ahmed WM, Nijsen TME, Goodacre R, Fowler SJ. Exhaled breath analysis: a review of ‘breath-taking’ methods for off-line analysis. Metabolomics. 2017;13:110.
145. Mutlu GM, Garey KW, Robbins RA, Danziger LH, Rubinstein I. Collection and analysis of exhaled breath condensate in humans. Am J Respir Crit Care Med. 2001;164:731-7.
146. Rahimpour E, Khoubnasabjafari M, Jouyban-Gharamaleki V, Jouyban A. Non-volatile compounds in exhaled breath condensate: review of methodological aspects. Anal Bioanal Chem. 2018;410:6411-40.
147. Carter SR, Davis CS, Kovacs EJ. Exhaled breath condensate collection in the mechanically ventilated patient. Respir Med. 2012;106:601-13.
148. Winters BR, Pleil JD, Angrish MM, Stiegel MA, Risby TH, Madden MC. Standardization of the collection of exhaled breath condensate and exhaled breath aerosol using a feedback regulated sampling device. J Breath Res. 2017;11:047107.
149. Shi M, Han W, Loudig O, et al. Initial development and testing of an exhaled microRNA detection strategy for lung cancer case-control discrimination. Sci Rep. 2023;13:6620.
150. Szunerits S, Dӧrfler H, Pagneux Q, et al. Exhaled breath condensate as bioanalyte: from collection considerations to biomarker sensing. Anal Bioanal Chem. 2023;415:27-34.
151. Half E, Ovcharenko A, Shmuel R, et al. Non-invasive multiple cancer screening using trained detection canines and artificial intelligence: a prospective double-blind study. Sci Rep. 2024;14:28204.
152. Patuleia SIS, van Gils CH, Oneto Cao AM, et al. The physiological microRNA landscape in nipple aspirate fluid: differences and similarities with breast tissue, breast milk, plasma and serum. Int J Mol Sci. 2020;21:8466.
153. Słyk-Gulewska P, Kondracka A, Kwaśniewska A. MicroRNA as a new bioactive component in breast milk. Noncoding RNA Res. 2023;8:520-6.
154. Jiwa N, Ezzat A, Holt J, Wijayatilake DS, Takats Z, Leff DR. Nipple aspirate fluid and its use for the early detection of breast cancer. Ann Med Surg. 2022;77:103625.
155. Patuleia SIS, van der Wall E, van Gils CH, et al. The changing microRNA landscape by color and cloudiness: a cautionary tale for nipple aspirate fluid biomarker analysis. Cell Oncol. 2021;44:1339-49.
156. Petrakis NL, Miike R, King EB, Lee L, Mason L, Chang-Lee B. Association of breast fluid coloration with age, ethnicity, and cigarette smoking. Breast Cancer Res Treat. 1988;11:255-62.
157. Csősz É, Emri G, Kalló G, Tsaprailis G, Tőzsér J. Highly abundant defense proteins in human sweat as revealed by targeted proteomics and label-free quantification mass spectrometry. J Eur Acad Dermatol Venereol. 2015;29:2024-31.
158. Harshman SW, Pitsch RL, Smith ZK, et al. The proteomic and metabolomic characterization of exercise-induced sweat for human performance monitoring: a pilot investigation. PLoS One. 2018;13:e0203133.
159. Maughan RJ, Shirreffs SM. Recovery from prolonged exercise: restoration of water and electrolyte balance. J Sports Sci. 1997;15:297-303.
160. Schittek B, Hipfel R, Sauer B, et al. Dermcidin: a novel human antibiotic peptide secreted by sweat glands. Nat Immunol. 2001;2:1133-7.
161. Cross T, Øvstebø R, Brusletto BS, et al. RNA profiles of tear fluid extracellular vesicles in patients with dry eye-related symptoms. Int J Mol Sci. 2023;24:15390.
162. Czarniak N, Kamińska J, Matowicka-Karna J, Koper-Lenkiewicz OM. Cerebrospinal fluid-basic concepts review. Biomedicines. 2023;11:1461.
163. Sandau US, Magaña SM, Costa J, et al.; International Society for Extracellular Vesicles Cerebrospinal Fluid Task Force. Recommendations for reproducibility of cerebrospinal fluid extracellular vesicle studies. J Extracell Vesicles. 2024;13:e12397.
164. Wichmann TO, Damkier HH, Pedersen M. A brief overview of the cerebrospinal fluid system and its implications for brain and spinal cord diseases. Front Hum Neurosci. 2021;15:737217.
165. Erdbrügger U, Blijdorp CJ, Bijnsdorp IV, et al. Urinary extracellular vesicles: a position paper by the Urine Task Force of the International Society for Extracellular Vesicles. J Extracell Vesicles. 2021;10:e12093.
166. Crescitelli R, Lässer C, Lötvall J. Isolation and characterization of extracellular vesicle subpopulations from tissues. Nat Protoc. 2021;16:1548-80.
167. Mincheva-Nilsson L, Baranov V, Nagaeva O, Dehlin E. Isolation and characterization of exosomes from cultures of tissue explants and cell lines. Curr Protoc Immunol. 2016;115:14.42.1-14.42.21.
168. Al-Humiari MA, Yu L, Liu LP, et al. Extracellular vesicles from BALF of pediatric cystic fibrosis and asthma patients increase epithelial sodium channel activity in small airway epithelial cells. Biochim Biophys Acta Biomembr. 2024;1866:184219.
169. Shaba E, Landi C, Carleo A, et al. Proteome characterization of BALF extracellular vesicles in idiopathic pulmonary fibrosis: unveiling undercover molecular pathways. Int J Mol Sci. 2021;22:5696.
170. Qin W, Zhang K, Sauter ER. Exosomal miRNAs in nipple aspirate fluid and breast cancer. Transl Cancer Res. 2017;6:S1304-10.
171. Bart G, Fischer D, Samoylenko A, et al. Characterization of nucleic acids from extracellular vesicle-enriched human sweat. BMC Genomics. 2021;22:425.
172. Herman S, Djaldetti R, Mollenhauer B, Offen D. CSF-derived extracellular vesicles from patients with Parkinson’s disease induce symptoms and pathology. Brain. 2023;146:209-24.
173. Yagi Y, Ohkubo T, Kawaji H, et al. Next-generation sequencing-based small RNA profiling of cerebrospinal fluid exosomes. Neurosci Lett. 2017;636:48-57.
174. Wijaya A, Julia V, Soedarsono N, et al. Comparative meta-analysis: salivary, plasma, and serum miRNA profiles for oral squamous cell carcinoma detection. J Pers Med. 2026;16:52.
175. Nobrega M, Reis MBD, Souza MF, et al. Comparative analysis of extracellular vesicles miRNAs (EV-miRNAs) and cell-free microRNAs (cf-miRNAs) reveals that EV-miRNAs are more promising as diagnostic and prognostic biomarkers for prostate cancer. Gene. 2025;939:149186.
176. He X, Park S, Chen Y, Lee H. Extracellular vesicle-associated miRNAs as a biomarker for lung cancer in liquid biopsy. Front Mol Biosci. 2021;8:630718.
177. Akers JC, Ramakrishnan V, Kim R, et al. MiR-21 in the extracellular vesicles (EVs) of cerebrospinal fluid (CSF): a platform for glioblastoma biomarker development. PLoS One. 2013;8:e78115.
178. Zhang Q, Jeppesen DK, Higginbotham JN, et al. Supermeres are functional extracellular nanoparticles replete with disease biomarkers and therapeutic targets. Nat Cell Biol. 2021;23:1240-54.
179. Zhang H, Freitas D, Kim HS, et al. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat Cell Biol. 2018;20:332-43.
180. Li P, Kaslan M, Lee SH, Yao J, Gao Z. Progress in exosome isolation techniques. Theranostics. 2017;7:789-804.
181. Suresh PS, Zhang Q. Comprehensive comparison of methods for isolation of extracellular vesicles from human plasma. J Proteome Res. 2025;24:2956-67.
182. Mrazova K, Henek T, Jurikova ZM, et al. Comparative evaluation of five extracellular vesicle isolation methods using proteomic profiling. AIMS Mol Sci. 2026;13:97-118.
183. Buschmann D, Kirchner B, Hermann S, et al. Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next-generation sequencing. J Extracell Vesicles. 2018;7:1481321.
184. Li WJ, Chen H, Tong ML, Niu JJ, Zhu XZ, Lin LR. Comparison of the yield and purity of plasma exosomes extracted by ultracentrifugation, precipitation, and membrane-based approaches. Open Chem. 2022;20:182-91.
185. Marqués-García F, Isidoro-García M. Protocols for exosome isolation and RNA profiling. Methods Mol Biol. 2016;1434:153-67.
186. Böing AN, van der Pol E, Grootemaat AE, Coumans FA, Sturk A, Nieuwland R. Single-step isolation of extracellular vesicles by size-exclusion chromatography. J Extracell Vesicles. 2014;3:23430.
187. Karimi N, Cvjetkovic A, Jang SC, et al. Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins. Cell Mol Life Sci. 2018;75:2873-86.
188. Guo J, Wu C, Lin X, et al. Establishment of a simplified dichotomic size-exclusion chromatography for isolating extracellular vesicles toward clinical applications. J Extracell Vesicles. 2021;10:e12145.
189. Monguió-Tortajada M, Morón-Font M, Gámez-Valero A, Carreras-Planella L, Borràs FE, Franquesa M. Extracellular-vesicle isolation from different biological fluids by size-exclusion chromatography. Curr Protoc Stem Cell Biol. 2019;49:e82.
190. Stranska R, Gysbrechts L, Wouters J, et al. Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma. J Transl Med. 2018;16:1.
191. Welton JL, Webber JP, Botos LA, Jones M, Clayton A. Ready-made chromatography columns for extracellular vesicle isolation from plasma. J Extracell Vesicles. 2015;4:27269.
192. Tóth EÁ, Turiák L, Visnovitz T, et al. Formation of a protein corona on the surface of extracellular vesicles in blood plasma. J Extracell Vesicles. 2021;10:e12140.
193. Wolf M, Poupardin RW, Ebner-Peking P, et al. A functional corona around extracellular vesicles enhances angiogenesis, skin regeneration and immunomodulation. J Extracell Vesicles. 2022;11:e12207.
194. Bergqvist M, Lässer C, Crescitelli R, Park KS, Lötvall J. A non-centrifugation method to concentrate and purify extracellular vesicles using superabsorbent polymer followed by size exclusion chromatography. J Extracell Vesicles. 2025;14:e70037.
195. Brennan K, Martin K, FitzGerald SP, et al. A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum. Sci Rep. 2020;10:1039.
196. Onódi Z, Pelyhe C, Terézia Nagy C, et al. Isolation of high-purity extracellular vesicles by the combination of iodixanol density gradient ultracentrifugation and bind-elute chromatography from blood plasma. Front Physiol. 2018;9:1479.
197. Iwai K, Minamisawa T, Suga K, Yajima Y, Shiba K. Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations. J Extracell Vesicles. 2016;5:30829.
198. Vaillancourt M, Hubert A, Subra C, et al. Velocity gradient separation reveals a new extracellular vesicle population enriched in miR-155 and mitochondrial DNA. Pathogens. 2021;10:526.
199. Ishida T, Hashimoto T, Masaki K, et al. Application of peptides with an affinity for phospholipid membranes during the automated purification of extracellular vesicles. Sci Rep. 2020;10:18718.
200. Masaki K, Ahmed ABF, Ishida T, et al. Chromatographic purification of small extracellular vesicles using an affinity column for phospholipid membranes. Biotechnol Lett. 2023;45:1457-66.
201. Sjoqvist S, Otake K, Hirozane Y. Analysis of cerebrospinal fluid extracellular vesicles by proximity extension assay: a comparative study of four isolation kits. Int J Mol Sci. 2020;21:9425.
202. Mathieu M, Martin-Jaular L, Lavieu G, Théry C. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nat Cell Biol. 2019;21:9-17.
203. Buzás EI, Tóth EÁ, Sódar BW, Szabó-Taylor KÉ. Molecular interactions at the surface of extracellular vesicles. Semin Immunopathol. 2018;40:453-64.
204. Konoshenko MY, Lekchnov EA, Vlassov AV, Laktionov PP. Isolation of extracellular vesicles: general methodologies and latest trends. Biomed Res Int. 2018;2018:8545347.
205. Mitchell MI, Ma J, Carter CL, Loudig O. Circulating exosome cargoes contain functionally diverse cancer biomarkers: from biogenesis and function to purification and potential translational utility. Cancers. 2022;14:3350.
206. Ströhle G, Gan J, Li H. Affinity-based isolation of extracellular vesicles and the effects on downstream molecular analysis. Anal Bioanal Chem. 2022;414:7051-67.
207. Shih CL, Chong KY, Hsu SC, et al. Development of a magnetic bead-based method for the collection of circulating extracellular vesicles. N Biotechnol. 2016;33:116-22.
208. Mitchell MI, Ben-Dov IZ, Liu C, et al. Extracellular vesicle capture by AnTibody of CHoice and Enzymatic Release (EV-CATCHER): a customizable purification assay designed for small-RNA biomarker identification and evaluation of circulating small-EVs. J Extracell Vesicles. 2021;10:e12110.
209. Benecke L, Chiang DM, Ebnoether E, Pfaffl MW, Muller L. Isolation and analysis of tumor-derived extracellular vesicles from head and neck squamous cell carcinoma plasma by galectin-based glycan recognition particles. Int J Oncol. 2022;61:133.
210. Chiang DM, Benecke L, Meng C, Ludwig C, Muller L, Pfaffl MW. Proteomic profiling of extracellular vesicles suggests Collectin10 as potential biomarker in relapsing head and neck squamous cell carcinoma. Trillium Extracell Vesicles. 2022.
211. Chen PC, Wu D, Hu CJ, Chen HY, Hsieh YC, Huang CC. Exosomal TAR DNA-binding protein-43 and neurofilaments in plasma of amyotrophic lateral sclerosis patients: a longitudinal follow-up study. J Neurol Sci. 2020;418:117070.
212. Zhang K, Yue Y, Wu S, Liu W, Shi J, Zhang Z. Rapid capture and nondestructive release of extracellular vesicles using aptamer-based magnetic isolation. ACS Sens. 2019;4:1245-51.
213. Nakai W, Yoshida T, Diez D, et al. A novel affinity-based method for the isolation of highly purified extracellular vesicles. Sci Rep. 2016;6:33935.
214. Kobayashi H, Shiba T, Yoshida T, et al. Precise analysis of single small extracellular vesicles using flow cytometry. Sci Rep. 2024;14:7465.
215. Kawakami K, Fujita Y, Kato T, et al. Diagnostic potential of serum extracellular vesicles expressing prostate-specific membrane antigen in urologic malignancies. Sci Rep. 2021;11:15000.
216. Khanabdali R, Mandrekar M, Grygiel R, et al. High-throughput surface epitope immunoaffinity isolation of extracellular vesicles and downstream analysis. Biol Methods Protoc. 2024;9:bpae032.
217. Reátegui E, van der Vos KE, Lai CP, et al. Engineered nanointerfaces for microfluidic isolation and molecular profiling of tumor-specific extracellular vesicles. Nat Commun. 2018;9:175.
218. Swatler J, Targońska A, Turos-Korgul L, Mosieniak G, Piwocka K. Protocol for isolation of tumor-derived extracellular vesicles and functional studies on human T cell subsets. STAR Protoc. 2024;5:103011.
219. Zhong W, Xiao Z, Qin Z, et al. Tumor-derived small extracellular vesicles inhibit the efficacy of CAR T cells against solid tumors. Cancer Res. 2023;83:2790-806.
220. Assarsson E, Lundberg M, Holmquist G, et al. Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability. PLoS One. 2014;9:e95192.
221. Gidlöf O, Evander M, Rezeli M, Marko-Varga G, Laurell T, Erlinge D. Proteomic profiling of extracellular vesicles reveals additional diagnostic biomarkers for myocardial infarction compared to plasma alone. Sci Rep. 2019;9:8991.
222. Fortunato D, Mladenović D, Criscuoli M, et al. Opportunities and pitfalls of fluorescent labeling methodologies for extracellular vesicle profiling on high-resolution single-particle platforms. Int J Mol Sci. 2021;22:10510.
223. Meggiolaro A, Moccia V, Brun P, et al. Microfluidic strategies for extracellular vesicle isolation: towards clinical applications. Biosensors. 2022;13:50.
224. Wang C, Qiu J, Liu M, et al. Microfluidic biochips for single-cell isolation and single-cell analysis of multiomics and exosomes. Adv Sci. 2024;11:e2401263.
225. Chen J, Zheng M, Xiao Q, et al. Recent advances in microfluidic-based extracellular vesicle analysis. Micromachines. 2024;15:630.
227. Welsh JA, Arkesteijn GJA, Bremer M, et al. A compendium of single extracellular vesicle flow cytometry. J Extracell Vesicles. 2023;12:e12299.
228. Szatanek R, Baj-Krzyworzeka M, Zimoch J, Lekka M, Siedlar M, Baran J. The methods of choice for extracellular vesicles (EVs) characterization. Int J Mol Sci. 2017;18:1153.
229. Görgens A, Corso G, Hagey DW, et al. Identification of storage conditions stabilizing extracellular vesicles preparations. J Extracell Vesicles. 2022;11:e12238.
230. Kuiper M, van de Nes A, Nieuwland R, Varga Z, van der Pol E. Reliable measurements of extracellular vesicles by clinical flow cytometry. Am J Reprod Immunol. 2021;85:e13350.
231. Libregts SFWM, Arkesteijn GJA, Németh A, Nolte-'t Hoen ENM, Wauben MHM. Flow cytometric analysis of extracellular vesicle subsets in plasma: impact of swarm by particles of non-interest. J Thromb Haemost. 2018;16:1423-36.
232. Cossarizza A, Chang HD, Radbruch A, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol. 2017;47:1584-797.
233. McGuinness CM, Grafstein B. Nucleolar changes in goldfish retinal ganglion cells in response to an optic nerve lesion are not perturbed by a second lesion. Exp Neurol. 1985;89:461-4.
234. Brealey J, Lees R, Tempest R, et al. Shining a light on fluorescent EV dyes: evaluating efficacy, specificity and suitability by nano-flow cytometry. J Extracell Biol. 2024;3:e70006.
235. Inglis HC, Danesh A, Shah A, Lacroix J, Spinella PC, Norris PJ. Techniques to improve detection and analysis of extracellular vesicles using flow cytometry. Cytometry A. 2015;87:1052-63.
236. Fletez-Brant K, Špidlen J, Brinkman RR, Roederer M, Chattopadhyay PK. flowClean: automated identification and removal of fluorescence anomalies in flow cytometry data. Cytometry A. 2016;89:461-71.
237. Tian Y, Gong M, Hu Y, et al. Quality and efficiency assessment of six extracellular vesicle isolation methods by nano-flow cytometry. J Extracell Vesicles. 2020;9:1697028.
238. Cao L, Wang H, Zhu J, et al. Exploring exosome profiling via CytoFLEX Nano flow cytometer: approaches and applications. VIEW. 2025;6:20250068.
239. Yim KHW, Krzyzaniak O, Al Hrout A, Peacock B, Chahwan R. Assessing extracellular vesicles in human biofluids using flow-based analyzers. Adv Healthc Mater. 2023;12:e2301706.
240. Groot Kormelink T, Arkesteijn GJ, Nauwelaers FA, van den Engh G, Nolte-'t Hoen EN, Wauben MH. Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry. Cytometry A. 2016;89:135-47.
241. Morales-Kastresana A, Musich TA, Welsh JA, et al. High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer. J Extracell Vesicles. 2019;8:1597603.
242. Pieragostino D, Lanuti P, Cicalini I, et al. Proteomics characterization of extracellular vesicles sorted by flow cytometry reveals a disease-specific molecular cross-talk from cerebrospinal fluid and tears in multiple sclerosis. J Proteomics. 2019;204:103403.
243. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-9.
244. Mack E, Neubauer A, Brendel C. Comparison of RNA yield from small cell populations sorted by flow cytometry applying different isolation procedures. Cytometry A. 2007;71:404-9.
245. Lima Â, Sousa LGV, Macedo F, Muzny CA, Cerca N. Assessing recovery rates of distinct exogenous controls for gDNA extraction efficiency using phenol-chloroform or silica-column based extractions. J Microbiol Methods. 2022;203:106607.
246. Wong RKY, MacMahon M, Woodside JV, Simpson DA. A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma. BMC Genomics. 2019;20:446.
247. Jiang L, Schlesinger F, Davis CA, et al. Synthetic spike-in standards for RNA-seq experiments. Genome Res. 2011;21:1543-51.
248. O’Grady T, Njock MS, Lion M, et al. Sorting and packaging of RNA into extracellular vesicles shape intracellular transcript levels. BMC Biol. 2022;20:72.
249. Turchinovich A, Drapkina O, Tonevitsky A. Transcriptome of extracellular vesicles: state-of-the-art. Front Immunol. 2019;10:202.
250. Dunlop RA, Banack SA, Cox PA. A comparison of the efficiency of RNA extraction from extracellular vesicles using the Qiagen RNeasy MinElute versus Enzymax LLC RNA Tini Spin columns and qPCR of miRNA. Biol Methods Protoc. 2021;6:bpab015.
251. Enderle D, Spiel A, Coticchia CM, et al. Characterization of RNA from exosomes and other extracellular vesicles isolated by a novel spin column-based method. PLoS One. 2015;10:e0136133.
252. Bryzgunova O, Konoshenko M, Zaporozhchenko I, Yakovlev A, Laktionov P. Isolation of cell-free miRNA from biological fluids: influencing factors and methods. Diagnostics. 2021;11:865.
253. Laurent LC, Abdel-Mageed AB, Adelson PD, et al. Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium. J Extracell Vesicles. 2015;4:26533.
254. Padilla JA, Barutcu S, Malet L, et al. Profiling the polyadenylated transcriptome of extracellular vesicles with long-read nanopore sequencing. BMC Genomics. 2023;24:564.
255. Scholes AN, Lewis JA. Comparison of RNA isolation methods on RNA-seq: implications for differential expression and meta-analyses. BMC Genomics. 2020;21:249.
256. Cabús L, Lagarde J, Curado J, Lizano E, Pérez-Boza J. Current challenges and best practices for cell-free long RNA biomarker discovery. Biomark Res. 2022;10:62.
257. Herbert ZT, Kershner JP, Butty VL, et al. Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction. BMC Genomics. 2018;19:199.
258. Thermo Fisher Scientific. NanoDrop spectrophotometer resources. Available from: https://www.thermofisher.com/de/de/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/uv-vis-spectrophotometry/instruments/nanodrop/resources.html#Documents-NanoDrop-Eight-Spectrophotometer. [Last accessed on 28 May 2026].
259. Thermo Fisher Scientific. NanoDrop micro-UV/Vis spectrophotometer - NanoDrop Eight - User Guide. Available from: https://documents.thermofisher.com/TFS-Assets/MSD/Product-Guides/M020-nanodrop-eight-spectrophotometer-user-guide.pdf. [Last accessed on 28 May 2026].
260. Okamoto T, Okabe S. Ultraviolet absorbance at 260 and 280 nm in RNA measurement is dependent on measurement solution. Int J Mol Med. 2000;5:657-9.
261. Hill AF, Pegtel DM, Lambertz U, et al. ISEV position paper: extracellular vesicle RNA analysis and bioinformatics. J Extracell Vesicles. 2013;2:22859.
262. Mateescu B, Kowal EJK, van Balkom BWM, et al. Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper. J Extracell Vesicles. 2017;6:1286095.
263. Benesova S, Kubista M, Valihrach L. Small RNA-sequencing: approaches and considerations for miRNA analysis. Diagnostics. 2021;11:964.
264. Buschmann D, Haberberger A, Kirchner B, et al. Toward reliable biomarker signatures in the age of liquid biopsies - how to standardize the small RNA-seq workflow. Nucleic Acids Res. 2016;44:5995-6018.
265. caRNAge. Comprehensive analysis of small RNA gene expression. Available from: https://www.mls.ls.tum.de/physio/forschung/carnage/. [Last accessed on 28 May 2026].
266. Rozowsky J, Kitchen RR, Park JJ, et al. exceRpt: a comprehensive analytic platform for extracellular RNA profiling. Cell Syst. 2019;8:352-7.e3.
267. Wiedrick JT, Phillips JI, Lusardi TA, et al. Validation of microRNA biomarkers for Alzheimer’s disease in human cerebrospinal fluid. J Alzheimers Dis. 2019;67:875-91.
268. Amarasinghe SL, Su S, Dong X, Zappia L, Ritchie ME, Gouil Q. Opportunities and challenges in long-read sequencing data analysis. Genome Biol. 2020;21:30.
269. Rafiq A, Kanavarioti A. The potential and limitations of the MinION/Yenos platform for miRNA-enabled early cancer detection. Int J Mol Sci. 2025;26:3822.
270. Shi C, Yang D, Ma X, et al. Quantitative and multiplexing analysis of microRNAs by direct full-length sequencing in nanopores. J Am Chem Soc. 2025;147:15614-24.
271. Zhang J, Yan S, Chang L, et al. Direct microRNA sequencing using nanopore-induced phase-shift sequencing. iScience. 2020;23:100916.
272. Crossland RE, Albiero A, Sanjurjo-Rodríguez C, et al. MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technology. J Extracell Biol. 2023;2:e72.
273. Daaboul GG, Gagni P, Benussi L, et al. Digital detection of exosomes by interferometric imaging. Sci Rep. 2016;6:37246.
274. Page GP, Zakharkin SO, Kim K, Mehta T, Chen L, Zhang K. Microarray analysis. Methods Mol Biol. 2007;404:409-30.
275. Johnson JM, Edwards S, Shoemaker D, Schadt EE. Dark matter in the genome: evidence of widespread transcription detected by microarray tiling experiments. Trends Genet. 2005;21:93-102.
276. Jaksik R, Iwanaszko M, Rzeszowska-Wolny J, Kimmel M. Microarray experiments and factors which affect their reliability. Biol Direct. 2015;10:46.
277. Geiss GK, Bumgarner RE, Birditt B, et al. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol. 2008;26:317-25.
278. Bustin SA. Improving the quality of quantitative polymerase chain reaction experiments: 15 years of MIQE. Mol Aspects Med. 2024;96:101249.
279. Huggett JF, Benes V, Bustin SA, et al. Cautionary note on contamination of reagents used for molecular detection of SARS-CoV-2. Clin Chem. 2020;66:1369-72.
280. Sequeira JP, Constâncio V, Salta S, et al. LiKidMiRs: a ddPCR-based panel of 4 circulating miRNAs for detection of renal cell carcinoma. Cancers. 2022;14:858.
281. dMIQE Group; Huggett JF. The digital MIQE guidelines update: minimum information for publication of quantitative digital PCR experiments for 2020. Clin Chem. 2020;66:1012-29.
282. Coumans FAW, Brisson AR, Buzas EI, et al. Methodological guidelines to study extracellular vesicles. Circ Res. 2017;120:1632-48.
283. Théry C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7:1535750.
284. Carpenter DK. Dynamic light scattering with applications to chemistry, biology, and physics (Berne, Bruce J.; Pecora, Robert). J Chem Educ. 1977;54:A430.
285. Cizmar P, Yuana Y. Detection and characterization of extracellular vesicles by transmission and cryo-transmission electron microscopy. Methods Mol Biol. 2017;1660:221-32.
286. Filipe V, Hawe A, Jiskoot W. Critical evaluation of nanoparticle tracking analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates. Pharm Res. 2010;27:796-810.
287. Kowal EJK, Ter-Ovanesyan D, Regev A, Church GM. Extracellular vesicle isolation and analysis by Western blotting. Methods Mol Biol. 2017;1660:143-52.
288. Buzas EI. Opportunities and challenges in studying the extracellular vesicle corona. Nat Cell Biol. 2022;24:1322-5.
289. Lötvall J, Hill AF, Hochberg F, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles. 2014;3:26913.
290. Van Deun J, Mestdagh P, Agostinis P, et al.; EV-TRACK Consortium. EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research. Nat Methods. 2017;14:228-32.
291. Poupardin R, Wolf M, Strunk D. Adherence to minimal experimental requirements for defining extracellular vesicles and their functions. Adv Drug Deliv Rev. 2021;176:113872.
292. Poupardin R, Wolf M, Maeding N, et al. Advances in extracellular vesicle research over the past decade: source and isolation method are connected with cargo and function. Adv Healthc Mater. 2024;13:e2303941.
293. Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611-22.
294. Ramsden SC, Daly S, Geilenkeuser WJ, et al. EQUAL-quant: an international external quality assessment scheme for real-time PCR. Clin Chem. 2006;52:1584-91.
295. Huggett JF, Foy CA, Benes V, et al. The digital MIQE guidelines: minimum information for publication of quantitative digital PCR experiments. Clin Chem. 2013;59:892-902.
296. Bustin SA, Ruijter JM, van den Hoff MJB, et al. MIQE 2.0: revision of the minimum information for publication of Quantitative Real-Time PCR Experiments Guidelines. Clin Chem. 2025;71:634-51.
297. Pfaffl MW, Kubista M, Vandesompele J, Bustin SA. MISEV and MIQE: integrating domain-specific and general standards to strengthen extracellular vesicle biomarker research. Extracell VesiclesCirc Nucl Acids. 2025;6:669-76.
298. ISEV. Scientific Reproducibility Task Forces. Available from: https://www.isev.org/task-forces. [Last accessed on 28 May 2026].
299. Dhondt B, Pinheiro C, Geeurickx E, et al. Benchmarking blood collection tubes and processing intervals for extracellular vesicle performance metrics. J Extracell Vesicles. 2023;12:e12315.
300. Ten-Doménech I, Ramos-Garcia V, Albiach-Delgado A, et al. Normalization approaches for extracellular vesicle-derived lipidomic fingerprints - a human milk case study. Chemom Intell Lab Syst. 2024;246:105070.
301. Blijdorp CJ, Tutakhel OAZ, Hartjes TA, et al. Comparing approaches to normalize, quantify, and characterize urinary extracellular vesicles. J Am Soc Nephrol. 2021;32:1210-26.
302. Vago R, Radano G, Zocco D, Zarovni N. Urine stabilization and normalization strategies favor unbiased analysis of urinary EV content. Sci Rep. 2022;12:17663.
303. Visnovitz T, Osteikoetxea X, Sódar BW, et al. An improved 96 well plate format lipid quantification assay for standardisation of experiments with extracellular vesicles. J Extracell Vesicles. 2019;8:1565263.
304. Wang C, Ding Q, Plant P, et al. Droplet digital PCR improves urinary exosomal miRNA detection compared to real-time PCR. Clin Biochem. 2019;67:54-9.
305. Brattelid T, Aarnes EK, Helgeland E, Guvaåg S, Eichele H, Jonassen AK. Normalization strategy is critical for the outcome of miRNA expression analyses in the rat heart. Physiol Genomics. 2011;43:604-10.
306. Latham GJ. Normalization of microRNA quantitative RT-PCR data in reduced scale experimental designs. Methods Mol Biol. 2010;667:19-31.
307. Andersen CL, Jensen JL, Ørntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004;64:5245-50.
308. Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:RESEARCH0034.
309. Luo M, Gao Z, Li H, et al. Selection of reference genes for miRNA qRT-PCR under abiotic stress in grapevine. Sci Rep. 2018;8:4444.
310. Ragni E, Perucca Orfei C, Silini AR, et al. miRNA reference genes in extracellular vesicles released from amniotic membrane-derived mesenchymal stromal cells. Pharmaceutics. 2020;12:347.
311. Pathak AK, Kural S, Kumar L, Saini S, Gupta M. Advances in algorithms for normalizer gene selection in qRT-PCR: implications for cancer biology and precision medicine. Front Genet. 2026;17:1762055.
312. Hildebrandt A, Kirchner B, Nolte-'t Hoen ENM, Pfaffl MW. miREV: an online database and tool to uncover potential reference RNAs and biomarkers in small-RNA sequencing data sets from extracellular vesicles enriched samples. J Mol Biol. 2021;433:167070.
313. Geeurickx E, Hendrix A. Targets, pitfalls and reference materials for liquid biopsy tests in cancer diagnostics. Mol Aspects Med. 2020;72:100828.
314. Geeurickx E, Tulkens J, Dhondt B, et al. The generation and use of recombinant extracellular vesicles as biological reference material. Nat Commun. 2019;10:3288.
315. Görgens A, Bremer M, Ferrer-Tur R, et al. Optimisation of imaging flow cytometry for the analysis of single extracellular vesicles by using fluorescence-tagged vesicles as biological reference material. J Extracell Vesicles. 2019;8:1587567.
316. Valkonen S, van der Pol E, Böing A, et al. Biological reference materials for extracellular vesicle studies. Eur J Pharm Sci. 2017;98:4-16.
317. Androvic P, Romanyuk N, Urdzikova-Machova L, Rohlova E, Kubista M, Valihrach L. Two-tailed RT-qPCR panel for quality control of circulating microRNA studies. Sci Rep. 2019;9:4255.
318. Jiang Z, Guo J, Xiao B, et al. Increased expression of miR-421 in human gastric carcinoma and its clinical association. J Gastroenterol. 2010;45:17-23.
319. Kachakova D, Mitkova A, Popov E, et al. Combinations of serum prostate-specific antigen and plasma expression levels of let-7c, miR-30c, miR-141, and miR-375 as potential better diagnostic biomarkers for prostate cancer. DNA Cell Biol. 2015;34:189-200.
320. Yaman Agaoglu F, Kovancilar M, Dizdar Y, et al. Investigation of miR-21, miR-141, and miR-221 in blood circulation of patients with prostate cancer. Tumour Biol. 2011;32:583-8.
321. Ibuki Y, Nishiyama Y, Tsutani Y, et al. Circulating microRNA/isomiRs as novel biomarkers of esophageal squamous cell carcinoma. PLoS One. 2020;15:e0231116.
322. Lim LP, Lau NC, Garrett-Engele P, et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature. 2005;433:769-73.
323. MiRXES. The genesis of GASTROClear. 2021. Available from: https://mirxes.com/the-genesis-of-gastroclear/. [Last accessed on 28 May 2026].
324. MiRXES. Mirxes receives FDA’s Breakthrough Device Designation for GASTROClear to advance blood-based cancer early detection. 2023. Available from: https://mirxes.com/mirxes-receives-fdas-breakthrough-device-designation-for-gastroclear-to-advance-blood-based-cancer-early-detection/. [Last accessed on 28 May 2026].
325. So JBY, Kapoor R, Zhu F, et al. Development and validation of a serum microRNA biomarker panel for detecting gastric cancer in a high-risk population. Gut. 2021;70:829-37.
326. Bianchi F, Nicassio F, Marzi M, et al. A serum circulating miRNA diagnostic test to identify asymptomatic high-risk individuals with early stage lung cancer. EMBO Mol Med. 2011;3:495-503.
327. Montani F, Marzi MJ, Dezi F, et al. miR-Test: a blood test for lung cancer early detection. J Natl Cancer Inst. 2015;107:djv063.
328. Mckay RJ. A graphical aid to selection of variables in two-group discriminant analysis. Applied Statistics. 1978;27:259.
329. Stacklies W, Redestig H, Scholz M, Walther D, Selbig J. pcaMethods--a bioconductor package providing PCA methods for incomplete data. Bioinformatics. 2007;23:1164-7.
330. Xu J, Wang W, Wang Y, et al. Progress in research on the role of exosomal miRNAs in the diagnosis and treatment of cardiovascular diseases. Front Genet. 2022;13:929231.
331. Yu Z, Saiki S, Shiina K, et al. Comprehensive data for studying serum exosome microRNA transcriptome in Parkinson’s disease patients. Sci Data. 2024;11:1128.
332. Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A. 1998;95:14863-8.
333. Pultar M, Oesterreicher J, Hartmann J, et al. Analysis of extracellular vesicle microRNA profiles reveals distinct blood and lymphatic endothelial cell origins. J Extracell Biol. 2024;3:e134.
335. Khalyfa A, Marin JM, Sanz-Rubio D, Lyu Z, Joshi T, Gozal D. Multi-omics analysis of circulating exosomes in adherent long-term treated OSA patients. Int J Mol Sci. 2023;24:16074.
336. Rohart F, Gautier B, Singh A, Lê Cao KA. mixOmics: an R package for 'omics feature selection and multiple data integration. PLoS Comput Biol. 2017;13:e1005752.
337. Fernandez-Valverde SL, Taft RJ, Mattick JS. Dynamic isomiR regulation in Drosophila development. RNA. 2010;16:1881-8.
338. Guo L, Liang T, Yu J, Zou Q. A comprehensive analysis of miRNA/isomiR expression with gender difference. PLoS One. 2016;11:e0154955.
339. Neilsen CT, Goodall GJ, Bracken CP. IsomiRs - the overlooked repertoire in the dynamic microRNAome. Trends Genet. 2012;28:544-9.
340. Makarenkov N, Yoel U, Haim Y, et al. Circulating isomiRs may be superior biomarkers compared to their corresponding miRNAs: a pilot biomarker study of using isomiR-ome to detect coronary calcium-based cardiovascular risk in patients with NAFLD. Int J Mol Sci. 2024;25:890.
341. Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 2005;33:e179.
342. Androvic P, Valihrach L, Elling J, Sjoback R, Kubista M. Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Res. 2017;45:e144.
343. Shi R, Chiang VL. Facile means for quantifying microRNA expression by real-time PCR. Biotechniques. 2005;39:519-25.
344. Raymond CK, Roberts BS, Garrett-Engele P, Lim LP, Johnson JM. Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA. 2005;11:1737-44.
345. Schmittgen TD, Jiang J, Liu Q, Yang L. A high-throughput method to monitor the expression of microRNA precursors. Nucleic Acids Res. 2004;32:e43.
346. Agarwal V, Bell GW, Nam JW, Bartel DP. Predicting effective microRNA target sites in mammalian mRNAs. Elife. 2015;4:e05005.
347. Chen Y, Wang X. miRDB: an online database for prediction of functional microRNA targets. Nucleic Acids Res. 2020;48:D127-31.
348. Hsu SD, Lin FM, Wu WY, et al. miRTarBase: a database curates experimentally validated microRNA-target interactions. Nucleic Acids Res. 2011;39:D163-9.
349. Xie GY, Song D, Luo T, Liao Y, Guo AY, Lei Q. EVmiRNA2.0: an updated database for miRNA expression in comprehensive human extracellular vesicles. Nucleic Acids Res. 2026;54:D128-34.
350. Ashburner M, Ball CA, Blake JA, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25-9.
351. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27-30.
352. Gillespie M, Jassal B, Stephan R, et al. The reactome pathway knowledgebase 2022. Nucleic Acids Res. 2022;50:D687-92.
353. Şen B, Che-Castaldo C, Akçakaya HR. The potential for species distribution models to distinguish source populations from sinks. J Anim Ecol. 2024;93:1924-34.
354. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102:15545-50.
355. Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16:284-7.
356. Puhka M, Thierens L, Nicorici D, et al. Exploration of extracellular vesicle miRNAs, targeted mRNAs and pathways in prostate cancer: relation to disease status and progression. Cancers. 2022;14:532.
357. Apeltrath C, Simon F, Riders A, Rudack C, Oberste M. Extracellular vesicle microRNAs as possible liquid biopsy markers in HNSCC-A longitudinal, monocentric study. Cancers. 2024;16:3793.
358. de Miguel Pérez D, Rodriguez Martínez A, Ortigosa Palomo A, et al. Extracellular vesicle-miRNAs as liquid biopsy biomarkers for disease identification and prognosis in metastatic colorectal cancer patients. Sci Rep. 2020;10:3974.
359. Freedman JE, Gerstein M, Mick E, et al. Diverse human extracellular RNAs are widely detected in human plasma. Nat Commun. 2016;7:11106.
360. Pfaffl MW. How laboratory guidelines promote the validity of circulating extracellular vesicle-associated nucleic acid biomarker signatures in liquid biopsy. Int J Mol Sci. 2025;26:12115.
361. Takizawa S, Matsuzaki J, Ochiya T. Circulating microRNAs: challenges with their use as liquid biopsy biomarkers. Cancer Biomark. 2022;35:1-9.
362. Tiberio P, Callari M, Angeloni V, Daidone MG, Appierto V. Challenges in using circulating miRNAs as cancer biomarkers. Biomed Res Int. 2015;2015:731479.







