Преаналитические особенности определения циркулирующих микроРНК как новых специфических биомаркеров реакции организма на физическую нагрузку

МикроРНК — малые некодирующие одноцепочечные РНК, длиной от 18 до 25 нуклеотидов, которые регулируют экспрессию генов на посттранскрипционном уровне посредством специфического связывания с мРНК-мишенью, приводящего к ее деградации. В последние десятилетия разработка технологий определения профилей экспрессии микроРНК стала важной частью исследовательских проектов, а роль микроРНК в качестве потенциальных высокоинформативных молекулярных биомаркеров различных физиологических и патологических процессов в организме активно изучается научным сообществом. В частности, физическая активность является важным модифицирующим фактором для циркулирующих микроРНК. В отличие от классических биохимических показателей крови, которые могут изменяться с течением времени в зависимости от температуры и условий хранения образца, микроРНК остаются стабильными при хранении и даже при многократных циклах замораживания-оттаивания, что делает их привлекательной и легкодоступной мишенью для обнаружения. Тем не менее определение профиля экспрессии микроРНК в клинической практике все еще является затруднительным из-за высокой неоднородности аналитических процедур, используемых для испытаний. В спортивной медицине особо важным является преаналитический этап, так как часто условия отбора биопроб не стандартизированы и могут влиять на результат анализа. В данном обзоре показана роль микроРНК в качестве новых чувствительных биомаркеров эффективности тренировочного процесса и регуляторов реакции организма в ответ на физическую активность, а также рассмотрены некоторые преаналитические аспекты анализа профилей экспрессии микроРНК.

1. Weber J.A., Baxter D.H., Zhang S., Huang D.Y., Huang K.H., Lee M.J., Galas D.J., Wang K. Themicro RNA spectrumin 12 body fluids. Clin. Chem. 2010;56(11):1733–1741. https://doi.org/10.1373/clinchem.2010.147405

2. Liang H., Gong F., Zhang S., Zhang C.Y., Zen K., Chen X. The origin, function, and diagnostic potential of extracellular microRNAs in human body fluids. Wiley Interdiscip. Rev. RNA. 2014; 5(2): 285–300. https://doi.org/10.1002/wrna.1208

3. Fazmin I. T., Achercouk Z., Edling C. E., Said A., Jeevaratnam K. Circulating microRNA as a Biomarker for Coronary Artery Disease. Biomolecules. 2020;10(10):1354. https://doi.org/10.3390/biom10101354

4. Mumford S.L., Towler B.P., Pashler A.L., Gilleard O., Martin Y., Newbury S.F. Circulating MicroRNA Biomarkers in Melanoma: Tools and Challenges in Personalised Medicine. Biomolecules. 2018;8(2):21. https://doi.org/10.3390/biom8020021

5. Butz H., Patócs A. MicroRNAs in endocrine tumors. EJIFCC. 2019;30(2):146–164.

6. do Amaral A.E., Cisilotto J., Creczynski-Pasa T.B., de Lucca Schiavon L. Circulating miRNAs in nontumoral liver diseases. Pharmacol. Res. 2018;128:274–287. https://doi.org/10.1016/j.phrs.2017.10.002

7. Felekkis K., Papaneophytou C. Challenges in Using Circulating Micro-RNAs as Biomarkers for Cardiovascular Diseases. Int. J. Mol. Sci. 2020;21(2):561. https://doi.org/10.3390/ijms21020561

8. Hüttenhofer A., Mayer G. Circulating miRNAs as biomarkers of kidney disease. Clin. Kidney J. 2017;10(1):27–29. https://doi.org/10.1093/ckj/sfw075

9. Grillari J., Mäkitie R. E., Kocijan R., Haschka J., Vázquez D.C., Semmelrock E., Hackl M. Circulating miRNAs in bone health and disease. Bone. 2021;145:115787. https://doi.org/10.1016/j.bone.2020.115787

10. Xie F., Liu Y.-L., Chen X.-Y., Li Q., Zhong J., Dai B.-Y., Shao X.-F., Wu G.-B. Role of MicroRNA, LncRNA, and Exosomes in the Progression of Osteoarthritis: A Review of Recent Literature. Orthop. Surg. 2020;12(3):708–716. https://doi.org/10.1111/os.12690

11. Tiwari A., Mukherjee B., Dixit M. MicroRNA Key to Angiogenesis Regulation: MiRNA Biology and Therapy. Curr. Cancer Drug Targets. 2018;18(3):266–277. https://doi.org/10.2174/1568009617666170630142725

12. Mahesh G., Biswas R. MicroRNA–155: A Master Regulator of Inflammation. J. Interferon. Cytokine Res. 2019;39(6):321–330. https://doi.org/10.1089/jir.2018.0155

13. Kamity R., Sharma S., Hanna N. MicroRNA–Mediated Control of Inflammation and Tolerance in Pregnancy. Front Immunol. 2019;10:718. https://doi.org/10.3389/fimmu.2019.00718

14. Pfaff N., Moritz T., Thum T., Cantz T. miRNAs involved in the generation, maintenance, and differentiation of pluripotent cells. J. Mol. Med. 2012;90(7):747–752. https://doi.org/10.1007/s00109-012-0922-z

15. Cheng A.M., Byrom M.W., Shelton J., Ford L.P. Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res. 2005;33(4):1290–1297. https://doi.org/10.1093/nar/gki200

16. Baggish A.L., Hale A., Weiner R.B., Lewis G.D., Systrom D., Wang F., Wang T.J., Chan S.Y. Dynamic regulation of circulating microRNA during acute exhaustive exercise and sustained aerobic exercise training. J. Physiol. 2011;589(Pt 16):3983–3994. https://doi.org/10.1113/jphysiol.2011.213363

17. Yin X., Cui S., Li X., Li W., Lu Q.J., Jiang X.H., Wang H., Chen X., Ma J.Z. Regulation of Circulatory Muscle–specific MicroRNA during 8 km Run. Int. J. Sports Med. 2020; 41(9):582–588. https://doi.org/10.1055/a-1145-3595

18. Caria A.C.I., Nonaka C.K, V., Pereira C.S., Soares M.B.P., Macambira S. G., de Freitas Souza B. S. Exercise Training–Induced Changes in MicroRNAs: Beneficial Regulatory Effects in Hypertension, Type 2 Diabetes, and Obesity. Int. J. Mol. Sci. 2018;19(11):3608. https://doi.org/10.3390/ijms19113608

19. Bandara K.V., Michael M.Z., Gleadle J.M. MicroRNA Biogenesis in Hypoxia. Microrna. 2017;6(2):80–96. https://doi.org/10.2174/2211536606666170313114821

20. Crosby M.E., Devlin C.M., Glazer P.M., Calin G.A., Ivan M. Emerging roles of microRNAs in the molecular responses to hypoxia. Curr. Pharm. Des. 2009;15(33):3861–3866. https://doi.org/10.2174/138161209789649367

21. Zhao J., Florentin J., Tai Y.-Y., Torrino S., Ohayon L., Brzoska T., et al. Long Range Endocrine Delivery of Circulating miR–210 to Endothelium Promotes Pulmonary Hypertension. Circ. Res. 2020;127(5):677–692. https://doi.org/10.1161/CIRCRESAHA.119.316398

22. Binderup H.G., Madsen J.S., Heegaard N.H.H., Houlind K., Andersen R.F., Brasen C.L. Quantification of microRNA levels in plasma — Impact of preanalytical and analytical conditions. PLoS One. 2018;13(7):e0201069. https://doi.org/10.1371/journal.pone.0201069

23. Lee R.C., Feinbaum R.L., Ambros V. The C. elegansheterochronic gene lin–4 encodessmall RNAs with antisense complementarity to lin-14. Cell. 1993;75(5):843–854. https://doi.org/10.1016/0092-8674(93)90529-y

24. Lagos-Quintana M., Rauhut R., Lendeckel W., Tuschl T. Identification of novel genescoding for small expressed RNAs. Science. 2001;294(5543):853–858. https://doi.org/10.1126/science.1064921

25. Reinhart B.J., Weinstein E.G., Rhoades M.W., Bartel B., Bartel D.P. MicroRNAs in plants. Genes Dev. 2002;16(13):1616–1626. https://doi.org/10.1101/gad.1004402

26. Grundhoff A., Sullivan C.S. Virus–encoded microRNAs. Virology. 2011;411(2):325–343. https://doi.org/10.1016/j.virol.2011.01.002

27. Griffiths-Jones S., Grocock R.J., van Dongen S., Bateman A., Enright A.J. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 2006;34:140–144. https://doi.org/10.1093/nar/gkj112

28. Kim V.N., Nam J.W. Genomics of microRNA. Trends Genet. 2006;22(3):165–173. https://doi.org/10.1016/j.tig.2006.01.003

29. Ghorai A., Ghosh U. miRNA gene counts in chromosomes vary widely in a speciesand biogenesis of miRNA largely depends on transcription or post–transcriptionalprocessing of coding genes. Front. Genet. 2014;5:100. https://doi.org/10.3389/fgene.2014.00100

30. Lujambio A., Calin G.A., Villanueva A., Ropero S., Sanchez-Cespedes M., Blanco D., et al. A microRNA DNA methylation signature for human cancer metastasis. Proc. Natl. Acad. Sci. U. S. A. 2008;105(36):13556–13561. https://doi.org/10.1073/pnas.0803055105

31. Hammond S.M. An overview of microRNAs. Adv. Drug Deliv. Rev. 2015;87:3–14. https://doi.org/10.1016/j.addr.2015.05.001

32. Bernstein E., Kim S.Y., Carmell M.A., Murchison E.P., Alcorn H., Li M.Z., et al. Dicer isessential for mouse development. Nat. Genet. 2003;35(3):215–217. https://doi.org/10.1038/ng1253

33. Suh M.R., Lee Y., Kim J.Y., Kim S.K., Moon S.H., Lee J.Y., et al. Human embryonicstem cells express a unique set of microRNAs. Dev. Biol. 2004;270(2):488–498. https://doi.org/10.1016/j.ydbio.2004.02.019

34. Murchison E.P., Partridge J.F., Tam O.H., Cheloufi S., Hannon G.J. Characterization ofDicer-deficient murine embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 2005;102(34):12135–12140. https://doi.org/10.1073/pnas.0505479102

35. Morrow D.A., de Lemos J.A. Benchmarks for the assessment of novel cardiovascular biomarkers. Circulation. 2007;115(8):949– 952. https://doi.org/10.1161/CIRCULATIONAHA.106.683110

36. Hackl M., Heilmeier U., Weilner S., Grillari J. Circulating microRNAs as novel biomarkersfor bone diseases — complex signatures for multifactorial diseases? Mol. Cell. Endocrinol. 2016;432:83–95. https://doi.org/10.1016/j.mce.2015.10.015

37. Mestdagh P., Hartmann N., Baeriswyl L., Andreasen D., Bernard N., Chen C., et al. Evaluation of quantitative miRNA expression platforms in the microRNA qualitycontrol (miRQC) study. Nat. Methods. 2014;11(8):809–815. https://doi.org/10.1038/nmeth.3014

38. Nelson P.T., Wang W.X., Wilfred B.R., Tang G. Technical variables in highthroughputmiRNA expression profiling: much work remains to be done. Biochim. Biophys. Acta. 2008;1779(11):758–765. https://doi.org/10.1016/j.bbagrm.2008.03.012

39. Chen X., Ba Y., Ma L., Cai X., Yin Y., Wang K., et al. Characterization ofmicroRNAsinserum: a novel class of biomarkers for diagnosis of cancer and other diseases. CellRes. 2008;18(10):997–1006. https://doi.org/10.1038/cr.2008.282

40. Takahashi K., Yokota S., Tatsumi N., Fukami T., Yokoi T., Nakajima M. Cigarettesmoking substantially alters plasma microRNA profiles in healthy subjects. Toxicol. Appl. Pharmacol. 2013;272(1):154–160. https://doi.org/10.1016/j.taap.2013.05.018

41. Witwer K.W. XenomiRs and miRNA homeostasis in health and disease: evidencethat diet and dietary miRNAs direct ly and indirectly influence circulating miRNAprofiles. RNA Biol. 2012;9(9):1147–1154. https://doi.org/10.4161/rna.21619

42. Shende V.R., Goldrick M.M., Ramani S., Earnest D.J. Expression and rhythmic modulationof circulating microRNAs targeting the clock gene Bmal1 inmice. PLoS One. 2011;6(7):e22586. https://doi.org/10.1371/journal.pone.0022586

43. Neal C.S., Michael M.Z., Pimlott L.K., Yong T.Y., Li J.Y., Gleadle J.M. Circulating microRNA expression is reduced in chronic kidney disease. Nephrol. Dial. Transplant. 2011;26(11):3794–3802. https://doi.org/10.1093/ndt/gfr485

44. Wang J., Chen J., Sen S. MicroRNA as biomarkers and diagnostics. J. Cell. Physiol. 2016;231(1):25–30. https://doi.org/10.1002/jcp.25056

45. Ardekani A.M., Naeini M.M. The role of microRNAs in human diseases. Avicenna J. Med. Biotechnol. 2010;2(4):161–179.

46. Lu J., Getz G., Miska E.A., Alvarez-Saavedra E., Lamb J., Peck D., Sweet-Cordero A., Ebert B. L., Mak R. H., Ferrando A. A., Downing J. R., Jacks T., Horvitz H. R., Golub T. R. MicroRNA expression profiles classify human cancers. Nature. 2005;435(7043):834–838. https://doi.org/10.1038/nature03702

47. Butz H., Patocs A. Technical aspects related to the analysis of circulating microRNAs. In: Circulating microRNAs in disease diagnostics and their potential biological relevance. Basel: Springer; 2015, p. 51–71. https://doi.org/10.1007/978-3-0348-0955-9_3

48. Lombardi G., Sanchis-Gomar F., Perego S., Sansoni V., Banfi G. Implications of exercise-induced adipo-myokines in bone metabolism. Endocrine. 2016;54(2):284–305. https://doi.org/10.1007/s12020-015-0834-0

49. Russell A.P., Hesselink M.K., Lo S.K., Schrauwen P. Regulation of metabolic transcriptional co-activators and transcription factors with acute exercise. FASEB J. 2005;19(8):986–988. https://doi.org/10.1096/fj.04-3168fje

50. Banfi G., Colombini A., Lombardi G., Lubkowska A. Metabolic markers in sports medicine. Adv. Clin. Chem. 2012;56:1–54. https://doi.org/10.1016/b978-0-12-394317-0.00015-7

51. Güller I., Russell A.P. MicroRNAs in skeletal muscle: their role and regulation in development,disease and function. J. Physiol. 2010;588(Pt 21):4075–4087. https://doi.org/10.1113/jphysiol.2010.194175

52. Ai J., Zhang R., Li Y., Pu J., Lu Y., Jiao J. Circulating microRNA– 1 as a potential novel biomarker for acute myocardial infarction. Biochem. Biophys. Res. Commun. 2010;391(1):73–77. https://doi.org/10.1016/j.bbrc.2009.11.005

53. McCarthy J.J., Esser K.A. MicroRNA–1 and microRNA– 133a expression are decreased during skeletal muscle hypertrophy. J. Appl. Physiol. 2007;102(1):306–313. https://doi.org/10.1152/japplphysiol.00932.2006

54. Wardle S.L, Bailey M.E.S., Kilikevicius A., Malkova D., Wilson R.H., Venckunas T., Moran C.N. Plasma microRNA levels differ between endurance and strength athletes. PLoS One. 2015;10(4):e0122107. https://doi.org/10.1371/journal.pone.0122107

55. Widmann M., Nieß A.M., Munz B. Physical Exercise and Epigenetic Modifications in Skeletal Muscle. Sports Med. 2019;49(4):509–523. https://doi.org/10.1007/s40279-019-01070-4

56. Polakovičová M., Musil P., Laczo E., Hamar D., Kyselovič J. Circulating MicroRNAs as Potential Biomarkers of Exercise Response. Int. J. Mol. Sci. 2016;17(10):1553. https://doi.org/10.3390/ijms17101553

57. Silva G.J.J., Bye A., El Azzouzi H., Wisløff U. MicroRNAs as Important Regulators of Exercise Adaptation. Prog. Cardiovasc. Dis. 2017;60(1):130–151. https://doi.org/10.1016/j.pcad.2017.06.003

58. Xu T., Liu Q., Yao J., Dai Y., Wang H., Xiao J. Circulating microRNAs in response to exercise. Scand. J. Med. Sci. Sports. 2015;25(2):149–154. https://doi.org/10.1111/sms.12421

59. Russell A.P., Lamon S. Exercise, skeletal muscle and circulating microRNAs. Prog.Mol. Biol. Transl. Sci. 2015;135:471–496. https://doi.org/10.1016/bs.pmbts.2015.07.018

60. Aoi W., Ichikawa H., Mune K., Tanimura Y., Mizushima K., Naito Y., Yoshikawa T. Muscle enriched microRNA miR– 486 decreases in circulation in response to exercise in young men. Front. Physiol. 2013;4:80. https://doi.org/10.3389/fphys.2013.00080

61. Nielsen S., Akerstrom T., Rinnov A., Yfanti C., Scheele C., Pedersen B.K., Laye M.J. The miRNA plasma signature in response to acute aerobic exercise and endurance training. PLoS One. 2014;9(2):e87308. https://doi.org/10.1371/journal.pone.0087308

62. Mooren F.C., Viereck J., Kruger K., Thum T. Circulating microRNAs as potential biomarkersof aerobic exercise capacity. Am. J. Physiol. Heart Circ. Physiol. 2014;306(4):H557–H563. https://doi.org/10.1152/ajpheart.00711.2013

63. Baggish A.L., Park J., Min P.K., Isaacs S., Parker B.A., Thompson P.D., et al. Rapid upregulation and clearance of distinct circulating microRNAs after prolonged aerobic exercise. J. Appl. Physiol. 2014;116(5):522–531. https://doi.org/10.1152/japplphysiol.01141.2013

64. Radom-Aizik S., Zaldivar Jr F., Leu S.Y., Adams G.R., Oliver S., Cooper D.M. Effects of exercise on microRNA expression in young males peripheral blood mononuclear cells. Clin. Transl. Sci. 2012;5(1):32–38. https://doi.org/10.1111/j.1752-8062.2011.00384.x

65. Drummond M.J., McCarthy J.J., Fry C.S., Esser K.A., Rasmussen B.B. Aging differentially affects human skeletal muscle microRNA expression at rest and after an anabolic stimulus of resistance exercise and essential amino acids. Am. J. Physiol. Endocrinol. Metab. 2008;295(6):E1333–E1340. https://doi.org/10.1152/ajpendo.90562.2008

66. Xu J., Lombardi G., Jiao W., Banfi G. Effects of exercise on bone status in female subjects, from young girls to postmenopausal women: an overview of systematic reviews and meta–analyses. Sports Med. 2016;46(8):1165–1182. https://doi.org/10.1007/s40279-016-0494-0

67. Qi Z., Liu W., Lu J. The mechanisms underlying the beneficial effects of exercise on bone remodeling: roles of bone–derived cytokines and microRNAs. Prog. Biophys. Mol. Biol. 2016;122(2):131–139. https://doi.org/10.1016/j.pbiomolbio.2016.05.010

68. Gamez B., Rodriguez-Carballo E., Ventura F. MicroRNAs and post-transcriptional regulation of skeletal development. J. Mol. Endocrinol. 2014;52(3):R179–R197. https://doi.org/10.1530/JME-13-0294

69. Lombardi G., Sansoni V., Perego S., Vernillo G., Bonzanni M., Merati G. Bone specific circulating miRNA profile changes over an 8-week repeated sprint training protocol. Endocr. Abstr. 2016;41:GP31. https://doi.org/10.1530/endoabs.41.GP31

70. Chen J., Qiu M., Dou C., Cao Z., Dong S. MicroRNAs in bone balance and osteoporosis. Drug Dev. Res. 2015;76(5):235–245. https://doi.org/10.1002/ddr.21260

71. Seeliger C., Karpinski K., Haug A.T., Vester H., Schmitt A., Bauer J.S., van Griensven M. Five freely circulating miRNAs and bone tissue miRNAs are associated with osteoporotic fractures. J. Bone Miner. Res. 2014;29(8):1718–1728. https://doi.org/10.1002/jbmr.2175

72. Rullman E., Mekjavic I.B., Fischer H., Eiken O. PlanHab (planetary habitat simulation): the combined and separate effects of 21 days bed rest and hypoxic confinement on human skeletal muscle miRNA expression. Physiol. Rep. 2016;4(8):e12753. https://doi.org/10.14814/phy2.12753

73. Bork-Jensen J., Scheele C., Christophersen D.V., Nilsson E., Friedrichsen M., Fernandez-Twinn D.S., et al. Glucose tolerance is associated with differential expression of microRNAs in skeletal muscle: results from studies of twins with and without type 2 diabetes. Diabetologia. 2015;58(2):363–373. https://doi.org/10.1007/s00125-014-3434-2

74. Alibegovic A.C., Sonne M.P., Hojbjerre L., Bork-Jensen J., Jacobsen S., Nilsson E., et al. Insulin resistance induced by physical inactivity is associated with multiple transcriptional changes in skeletal muscle in young men. Am. J. Physiol. Endocrinol. Metab. 2010;299(5):E752–E763. https://doi.org/10.1152/ajpendo.00590.2009

75. Gallagher I.J., Scheele C., Keller P., Nielsen A.R., Remenyi J., Fischer C.P., et al. Integration of microRNA changes in vivo identifies novel molecular features of muscle insulin resistance in type 2 diabetes. Genome Med. 2010;2(2):9. https://doi.org/10.1186/gm130

76. Wahlquist C., Jeong D., Rojas-Munoz A., Kho C., Lee A., Mitsuyama S., et al. Inhibition of miR–25 improves cardiac contractility in the failing heart. Nature. 2014;508(7497):531–535. https://doi.org/10.1038/nature13073

77. Becker N., Lockwood C.M. Pre-analytical variables in miRNA analysis. Clin. Biochem. 2013;46(10-11):861–868. https://doi.org/10.1016/j.clinbiochem.2013.02.015

78. Lombardi G., Lanteri P., Colombini A., Banfi G. Blood biochemical markers of bone turnover: pre–analytical and technical aspects of sample collection and handling. Clin. Chem. Lab. Med. 2012;50(5):771–789. https://doi.org/10.1515/cclm-2011-0614

79. Mestdagh P., Van Vlierberghe P., DeWeer A., Muth D., Westermann F., Speleman F. Vandesompele J. A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol. 2009;10(6):R64. https://doi.org/10.1186/gb-2009-10-6-r64

80. Bonini P., Plebani M., Ceriotti F., Rubboli F. Errors in laboratory medicine. Clin. Chem. 2002;48(5):691–698.

81. Lippi G., Guidi G.C., Mattiuzzi C., Plebani M. Preanalytical variability: the darkside of the moon in laboratory testing. Clin. Chem. Lab. Med. 2006;44(4):358–365. https://doi.org/10.1515/CCLM.2006.073

82. Kavsak P.A. What is in that sample? A pertinent question when assessing quality for patient laboratory results and beyond. Clin. Biochem. 2015;48(7-8):465–466. https://doi.org/10.1016/j.clinbiochem.2015.04.010

83. van Dongen-Lases E.C., Cornes M.P., Grankvist K., Ibarz M., Kristensen G.B., Lippi G., et al. Patient identification and tube labelling — a call for harmonization. Clin. Chem. Lab. Med. 2016;54(7):1141–1145. https://doi.org/10.1515/cclm-2015-1089

84. Lippi G., Chance J.J., Church S., Dazzi P., Fontana R., Giavarina D., et al. Preanalytical quality improvement: from dream to reality. Clin. Chem. Lab. Med. 2011;49(7):1113–1126. https://doi.org/10.1515/CCLM.2011.600

85. Supak-Smolcic V., Antoncic D., Ozanic D., Vladilo I., Bilic-Zulle L. Influence of a prolonged fasting and mild activity on routine laboratory tests. Clin. Biochem. 2015;48(1-2):85–88. https://doi.org/10.1016/j.clinbiochem.2014.10.005

86. Banfi G., Dolci A. Preanalytical phase of sport biochemistry and haematology. J. Sports Med. Phys. Fitness. 2003;43(2):223–230.

87. Lima-Oliveira G., Guidi G.C., Salvagno G.L., Brocco G., Danese E., Lippi G. Estimation of the imprecision on clinical chemistry testing due to fist clenchingand maintenance during venipuncture. Clin. Biochem. 2016;49(18):1364–1367. https://doi.org/10.1016/j.clinbiochem.2016.07.007

88. Bahtiyar N., Yoldas A., Abbak Y., Dariyerli N., Toplan S. Erythroid microRNA and oxidant status alterations in l–thyroxine– induced hyperthyroid rats: effects of selenium supplementation. Minerva Endocrinol. (Torino). 2021;46(1):107–115. https://doi.org/10.23736/S2724-6507.20.03154-5

89. Quality Venipuncture Quick Guide Standard by Clinical and Laboratory Standards Institute [Internet]. Available from: https://www.techstreet.com/standards/clsi-h03-a6-quickguide?product_id=1732666#jumps

90. Lima-Oliveira G., Lippi G., Salvagno G.L., Montagnana M., Picheth G., Guidi G.C. The effective reduction of tourniquet application time after minor modification of the CLSI H03–A6 blood collection procedure. Biochem. Med. 2013;23(3):308–315. https://doi.org/10.11613/bm.2013.037

91. WADA, The world anti-doping code: athlete biological passport operating guidelines and compilation of required elements [Internet]. Available from: https://www.wada-ama.org/sites/default/files/resources/files/guidelines_abp_v8_final.pdf

92. Lombardi G., Perego S., Luzi L., Banfi G. A four-season molecule: osteocalcin. Updates in its physiological roles. Endocrine. 2015;48(2):394–404. https://doi.org/10.1007/s12020-014-0401-0

93. Lanteri P., Lombardi G., Colombini A., Banfi G. Vitamin D in exercise: physiologic and analytical concerns. Clin. Chim. Acta. 2013;415:45–53. https://doi.org/10.1016/j.cca.2012.09.004

94. Lombardi G., Banfi G. Effects of sample matrix and storage conditions on fulllengthvisfatin measurement in blood. Clin. Chim. Acta. 2015;440:140–142. https://doi.org/10.1016/j.cca.2014.11.006

95. Lanteri P., Lombardi G., Colombini A., Grasso D., Banfi G. Stability of osteopontin in plasma and serum. Clin. Chem. Lab. Med. 2012;50(11):1979–1984. https://doi.org/10.1515/cclm-2012-0177

96. Segura J., Lundby C. Blood doping: potential of blood and urine sampling to detect autologous transfusion. Br. J. Sports Med. 2014;48(10):837–841. https://doi.org/10.1136/bjsports-2014-093601

97. Lombardi G., Colombini A., Lanteri P., Banfi G. Reticulocytes in sports medicine: an update, Adv. Clin. Chem. 2013;59:125–153. https://doi.org/10.1016/b978-0-12-405211-6.00005-x

98. Banfi G., Lombardi G., Colombini A., Lippi G. Analytical variability in sport hematology:its importance in an antidoping setting. Clin. Chem. Lab. Med. 2011;49(5):779–782. https://doi.org/10.1515/CCLM.2011.125

99. Jarry J., Schadendorf D., Greenwood C., Spatz A., van Kempen L.C. The validity of circulating microRNAs in oncology: five years of challenges and contradictions. Mol. Oncol. 2014;8(4):819–829. https://doi.org/10.1016/j.molonc.2014.02.009

100. Chen X., Liang H., Zhang J., Zen K., Zhang C.Y. Secreted microRNAs: a new form of intercellular communication. Trends Cell Biol. 2012;22(3):125–132. https://doi.org/10.1016/j.tcb.2011.12.001

101. Arroyo J.D., Chevillet J.R., Kroh E.M., Ruf I.K., Pritchard C.C., Gibson D.F. Argonaute2 complexes carry a population of circulating microRNAs independentof vesicles in human plasma. Proc. Natl. Acad. Sci. U. S. A. 2011;108(12):5003–5008. https://doi.org/10.1073/pnas.1019055108

102. Li L., Zhu D., Huang L., Zhang J., Bian Z., Chen X., et al. Argonaute 2 complexesselectively protect the circulating microRNAs in cell–secreted microvesicles. PLoSOne. 2012;7(10):e46957. https://doi.org/10.1371/journal.pone.0046957103.

103. El-Hefnawy T., Raja S., Kelly L., Bigbee W.L., Kirkwood J.M., Luketich J.D. Characterization of amplifiable, circulating RNA in plasma and its potential as atool for cancer diagnostics. Clin. Chem. 2004;50(3):564–573. https://doi.org/10.1373/clinchem.2003.028506

104. Mitchell P.S., Parkin R.K., Kroh E.M., Fritz B.R., Wyman S.K., Pogosova-Agadjanyan E.L., et al. Circulating microRNAs as stable blood–basedmarkers for cancer detection. Proc. Natl. Acad. Sci. U. S. A. 2008;105(30):10513–10518. https://doi.org/10.1073/pnas.0804549105

105. Kroh E.M., Parkin R.K., Mitchell P.S., Tewari M. Analysis of circulating microRNAbiomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR). Methods. 2010;50(4):298–301. https://doi.org/10.1016/j.ymeth.2010.01.032

106. Cheng H.H., Yi H.S., Kim Y., Kroh E.M., Chien J.W., Eaton K.D., et al. Plasmaprocessing conditions substantially influence circulating microRNA biomarker levels. PLoS One. 2013;8(6):e64795. https://doi.org/10.1371/journal.pone.0064795

107. Kirschner M.B., Kao S.C., Edelman J.J., Armstrong N.J., Vallely M.P., van Zandwijk N., Reid G. Haemolysis during sample preparation alters microRNA content of plasma. PLoS One. 2011;6(9):e24145. https://doi.org/10.1371/journal.pone.0024145

108. Willeit P., Zampetaki A., Dudek K., Kaudewitz D., King A., Kirkby N.S., et al. Circulating microRNAs as novel biomarkers for platelet activation. Circ. Res. 2013;112(4):595–600. https://doi.org/10.1161/CIRCRESAHA.111.300539

109. Blondal T., Jensby Nielsen S., Baker A., Andreasen D., Mouritzen P., Wrang Teilum M., Dahlsveen I.K. Assessing sample and miRNA profile quality in serum and plasma or other biofluids. Methods. 2013;59(1):S1–S6. https://doi.org/10.1016/j.ymeth.2012.09.015

110. Livesey J.H., Ellis M.J., Evans M.J. Pre-analytical requirements. Clin. Biochem. Rev. 2008;29(1):S11–S15.

111. Kavsak P.A., Hammett-Stabler C.A. Clinical biochemistry year in review — theclinical “good”, the analytical “bad”, and the “ugly” laboratory practices. Clin. Biochem. 2014;47(18):255–256. https://doi.org/10.1016/j.clinbiochem.2014.11.015

112. Boeckel J.N., Thome C.E., Leistner D., Zeiher A.M., Fichtlscherer S., Dimmeler S. Heparin selectively affects the quantification of microRNAs in human bloodsamples. Clin. Chem. 2013;59(7):1125–1127. https://doi.org/10.1373/clinchem.2012.199505

113. Garcia M.E., Blanco J.L., Caballero J., Gargallo-Viola D. Anticoagulants interferewith PCR used to diagnose invasive aspergillosis. J. Clin. Microbiol. 2002;40(4):1567–1568. https://doi.org/10.1128/JCM.40.4.1567-1568.2002

114. Zampetaki A., Mayr M. Analytical challenges and technical limitations in assessing circulating miRNAs. Thromb. Haemost. 2012;108(4):592–598. https://doi.org/10.1160/TH12-02-0097

115. Kim D.J., Linnstaedt S., Palma J., Park J.C., Ntrivalas E., Kwak-Kim J.Y., et al. Plasma components affect accuracy of circulating cancer–related microRNA quantitation. J. Mol. Diagn. 2012;14(1):71–80. https://doi.org/10.1016/j.jmoldx.2011.09.002

116. Lombardi G., Perego S., Sansoni V., Banfi G. Circulating miRNA as fine regulators of the physiological responses to physical activity: Pre-analytical warnings for a novel class of biomarkers. Clin. Biochem. 2016;49(18):1331–1339. https://doi.org/10.1016/j.clinbiochem.2016.09.017

117. Turchinovich A., Weiz L., Langheinz A., Burwinkel B. Characterization ofextracellular circulating microRNA. Nucleic Acids Res. 2011;39(16):7223–7233. https://doi.org/10.1093/nar/gkr254

118. Guimbellot J.S., Erickson S.W., Mehta T., Wen H., Page G.P., Sorscher E.J., Hong J. S. Correlation of microRNA levels during hypoxia with predicted target mRNAs through genome – wide microarray analysis. BMC Med. Genet. 2009;2:15. https://doi.org/10.1186/1755-8794-2-15

119. Chen F., Zhang W., Liang Y., Huang J., Li K., Green C.D., et al. Transcriptome and network changes in climbers at extreme altitudes. PLoS One. 2012;7(2):e31645. https://doi.org/10.1371/journal.pone.0031645

120. Yan Y., Shi Y., Wang C., Guo P., Wang J., Zhang C.Y., Zhang C. Influence of a high altitude hypoxic environment on human plasma microRNA profiles. Sci. Rep. 2015;5:15156. https://doi.org/10.1038/srep15156