Technical Aspects Related to the Analysis of Circulating microRNAs

  • Henriett Butz
  • Attila PatócsEmail author
Part of the Experientia Supplementum book series (EXS, volume 106)


Specific and sensitive noninvasive biofluid-based biomarkers are always needed in the laboratory diagnosis of diseases. Biomarkers are applied not only for diagnostic purposes but for stratifying a disease and for assessing the therapy response or disease progression. MicroRNAs (miRNAs) are short noncoding RNA molecules regulating gene expression posttranscriptionally. They are frequently dysregulated in many physiological and pathophysiological conditions. miRNAs are present in the circulation and in other biofluids that are common matrices for clinical laboratory testing that has raised the possibility that miRNAs may serve as novel biomarkers. Their excellent stability also supports the possibility that miRNAs once will be routinely used biomarkers in clinical practice. From an analytical point of view, there are many factors (starting material, sample storage and processing, different RNA extraction and detection methods, intra- and interassay variability, and assay interferences) to consider if a miRNA as biomarker is aimed to be introduced as a clinical laboratory test. Despite several pre-analytical and analytical factors that still need standardization, a significant number of studies have been published about the potential role of circulating miRNAs as biomarkers. Due to the lack of standardization of methods, there are a lot of discrepancies among results. In this chapter, we aimed to summarize the current findings about circulating miRNAs focusing on the analytical points related to miRNAs measurements from biofluids.


miRNA Biomarker Laboratory test Technical issues Biofluid 


  1. Akane A, Matsubara K, Nakamura H et al (1994) Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. J Forensic Sci 39:362–372CrossRefPubMedGoogle Scholar
  2. Al-Soud WA, Rådström P (2001) Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 39:485–493CrossRefPubMedPubMedCentralGoogle Scholar
  3. Al-Soud WA, Jönsson LJ, Râdström P (2000) Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol 38:345–350PubMedPubMedCentralGoogle Scholar
  4. Arroyo JD, Chevillet JR, Kroh EM et al (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci USA 108:5003–5008CrossRefPubMedPubMedCentralGoogle Scholar
  5. Benz F, Roderburg C, Vargas Cardenas D et al (2013) U6 is unsuitable for normalization of serum miRNA levels in patients with sepsis or liver fibrosis. Exp Mol Med 45, e42CrossRefPubMedPubMedCentralGoogle Scholar
  6. Brase JC, Wuttig D, Kuner R et al (2010) Serum microRNAs as non-invasive biomarkers for cancer. Mol Cancer 9:306CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bryant RJ, Pawlowski T, Catto JW et al (2012) Changes in circulating microRNA levels associated with prostate cancer. Br J Cancer 106:768–774CrossRefPubMedPubMedCentralGoogle Scholar
  8. Castoldi M, Schmidt S, Benes V et al (2006) A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA). RNA 12:913–920CrossRefPubMedPubMedCentralGoogle Scholar
  9. Chen X, Ba Y, Ma L et al (2008) Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 18:997–1006CrossRefPubMedGoogle Scholar
  10. Cheng G (2015) Circulating miRNAs: roles in cancer diagnosis, prognosis and therapy. Adv Drug Deliv Rev 81:75–93CrossRefPubMedGoogle Scholar
  11. Cheng Y, Zhang X, Li Z et al (2009) Highly sensitive determination of microRNA using target-primed and branched rolling-circle amplification. Angew Chem Int Ed Engl 48:3268–3272CrossRefPubMedGoogle Scholar
  12. Chevillet JR, Lee I, Briggs HA et al (2014) Issues and prospects of microRNA-based biomarkers in blood and other body fluids. Molecules 19:6080–6105CrossRefPubMedGoogle Scholar
  13. Cissell KA, Rahimi Y, Shrestha S et al (2008) Bioluminescence-based detection of microRNA, miR21 in breast cancer cells. Anal Chem 80:2319–2325CrossRefPubMedGoogle Scholar
  14. Cortez MA, Bueso-Ramos C, Ferdin J et al (2011) MicroRNAs in body fluids--the mix of hormones and biomarkers. Nat Rev Clin Oncol 8:467–477CrossRefPubMedPubMedCentralGoogle Scholar
  15. Duygu B, de Windt LJ, da Costa Martins PA (2015) Targeting microRNAs in heart failure. Trends Cardiovasc Med. doi: 10.1016/j.tcm.2015.05.008 [Epub ahead of print]PubMedGoogle Scholar
  16. Farazi TA, Hoell JI, Morozov P et al (2013) MicroRNAs in human cancer. Adv Exp Med Biol 774:1–20CrossRefPubMedPubMedCentralGoogle Scholar
  17. Farina NH, Wood ME, Perrapato SD et al (2014) Standardizing analysis of circulating microRNA: clinical and biological relevance. J Cell Biochem 115:805–811CrossRefPubMedPubMedCentralGoogle Scholar
  18. Gadelha MR, Kasuki L, Dénes J et al (2013) MicroRNAs: suggested role in pituitary adenoma pathogenesis. J Endocrinol Invest 36:889–895CrossRefPubMedGoogle Scholar
  19. Git A, Dvinge H, Salmon-Divon M et al (2010) Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microRNA expression. RNA 16:991–1006CrossRefPubMedPubMedCentralGoogle Scholar
  20. Grasedieck S, Sorrentino A, Langer C et al (2013) Circulating microRNAs in hematological diseases: principles, challenges, and perspectives. Blood 121:4977–4984CrossRefPubMedGoogle Scholar
  21. Heegaard NH, Schetter AJ, Welsh JA et al (2012) Circulating micro-RNA expression profiles in early stage nonsmall cell lung cancer. Int J Cancer 130:1378–1386CrossRefPubMedGoogle Scholar
  22. Hindson CM, Chevillet JR, Briggs HA et al (2013) Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 10:1003–1005CrossRefPubMedPubMedCentralGoogle Scholar
  23. Hong CY, Chen X, Liu T et al (2013) Ultrasensitive electrochemical detection of cancer-associated circulating microRNA in serum samples based on DNA concatamers. Biosens Bioelectron 50:132–136CrossRefPubMedGoogle Scholar
  24. Hossain MM, Sohel MM, Schellander K et al (2012) Characterization and importance of microRNAs in mammalian gonadal functions. Cell Tissue Res 349:679–690CrossRefPubMedGoogle Scholar
  25. Hu J, Wang Z, Liao BY et al (2014) Human miR-1228 as a stable endogenous control for the quantification of circulating microRNAs in cancer patients. Int J Cancer 135:1187–1194CrossRefPubMedGoogle Scholar
  26. Huang X, Yuan T, Tschannen M et al (2013) Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC Genomics 14:319CrossRefPubMedPubMedCentralGoogle Scholar
  27. Jia H, Li Z, Liu C et al (2010) Ultrasensitive detection of microRNAs by exponential isothermal amplification. Angew Chem Int Ed Engl 49:5498–5501CrossRefPubMedGoogle Scholar
  28. Jiang Z, Song Q, Yang S et al (2015) Serum microRNA-218 is a potential biomarker for esophageal cancer. Cancer Biomark. doi: 10.3233/CBM-150480 [Epub ahead of print]Google Scholar
  29. Kim DJ, Linnstaedt S, Palma J et al (2012a) Plasma components affect accuracy of circulating cancer-related microRNA quantitation. J Mol Diagn 14:71–80CrossRefPubMedPubMedCentralGoogle Scholar
  30. Kim YK, Yeo J, Kim B et al (2012b) Short structured RNAs with low GC content are selectively lost during extraction from a small number of cells. Mol Cell 46:893–895CrossRefPubMedGoogle Scholar
  31. Köberle V, Pleli T, Schmithals C et al (2013) Differential stability of cell-free circulating microRNAs: implications for their utilization as biomarkers. PLoS ONE 8, e75184CrossRefPubMedPubMedCentralGoogle Scholar
  32. Kriebel S, Schmidt D, Holdenrieder S et al (2015) Analysis of tissue and serum microRNA expression in patients with upper urinary tract urothelial cancer. PLoS ONE 10, e0117284CrossRefPubMedPubMedCentralGoogle Scholar
  33. Lagos-Quintana M, Rauhut R, Lendeckel W et al (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853–858CrossRefPubMedGoogle Scholar
  34. Lawrie CH, Gal S, Dunlop HM et al (2008) Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br J Haematol 141:672–675CrossRefPubMedGoogle Scholar
  35. Leone V, D’Angelo D, Ferraro A et al (2011) A TSH-CREB1-microRNA loop is required for thyroid cell growth. Mol Endocrinol 10:1819–1830CrossRefGoogle Scholar
  36. Li X (2014) MiR-375, a microRNA related to diabetes. Gene 533:1–4CrossRefPubMedGoogle Scholar
  37. Li A, Yu J, Kim H et al (2013) MicroRNA array analysis finds elevated serum miR-1290 accurately distinguishes patients with low-stage pancreatic cancer from healthy and disease controls. Clin Cancer Res 19:3600–3610CrossRefPubMedPubMedCentralGoogle Scholar
  38. Lisse TS, Adams JS, Hewison M (2013) Vitamin D and microRNAs in bone. Crit Rev Eukaryot Gene Expr 23:195–214CrossRefPubMedPubMedCentralGoogle Scholar
  39. McDonald JS, Milosevic D, Reddi HV et al (2011) Analysis of circulating microRNA: preanalytical and analytical challenges. Clin Chem 57:833–840CrossRefPubMedGoogle Scholar
  40. Mestdagh P, Hartmann N, Baeriswyl L et al (2014) Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study. Nat Methods 11:809–815CrossRefPubMedGoogle Scholar
  41. Meyer SU, Pfaffl MW, Ulbrich SE (2010) Normalization strategies for microRNA profiling experiments: a ‘normal’ way to a hidden layer of complexity? Biotechnol Lett 32:1777–1788CrossRefPubMedGoogle Scholar
  42. Mishra PJ, Bertino JR (2009) MicroRNA polymorphisms: the future of pharmacogenomics, molecular epidemiology and individualized medicine. Pharmacogenomics 10:399–416CrossRefPubMedPubMedCentralGoogle Scholar
  43. Mishra PJ, Merlino G (2009) MicroRNA reexpression as differentiation therapy in cancer. J Clin Invest 119:2119–2123PubMedPubMedCentralGoogle Scholar
  44. Mishra PJ, Humeniuk R, Mishra PJ et al (2007) A miR-24 microRNA binding-site polymorphism in dihydrofolate reductase gene leads to methotrexate resistance. Proc Natl Acad Sci USA 104:13513–13518CrossRefPubMedPubMedCentralGoogle Scholar
  45. Mishra PJ, Song B, Mishra PJ et al (2009) MiR-24 tumor suppressor activity is regulated independent of p53 and through a target site polymorphism. PLoS ONE 4, e8445CrossRefPubMedPubMedCentralGoogle Scholar
  46. Mitchell PS, Parkin RK, Kroh EM et al (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 105:10513–10518CrossRefPubMedPubMedCentralGoogle Scholar
  47. Nelson PT, Baldwin DA, Scearce LM et al (2004) Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods 1:155–161CrossRefPubMedGoogle Scholar
  48. Orom UA, Nielsen FC, Lund AH (2008) MicroRNA-10a binds the 5′UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell 30:460–471CrossRefPubMedGoogle Scholar
  49. Page K, Guttery DS, Zahra N et al (2013) Influence of plasma processing on recovery and analysis of circulating nucleic acids. PLoS ONE 8, e77963CrossRefPubMedPubMedCentralGoogle Scholar
  50. Pallante P, Battista S, Pierantoni GM et al (2014) Deregulation of microRNA expression in thyroid neoplasias. Nat Rev Endocrinol 10:88–101CrossRefPubMedGoogle Scholar
  51. Place RF, Li LC, Pookot D et al (2008) MicroRNA-373 induces expression of genes with complementary promoter sequences. Proc Natl Acad Sci USA 105:1608–1613CrossRefPubMedPubMedCentralGoogle Scholar
  52. Pritchard CC, Cheng HH, Tewari M (2012a) MicroRNA profiling: approaches and considerations. Nat Rev Genet 13:358–369CrossRefPubMedPubMedCentralGoogle Scholar
  53. Pritchard CC, Kroh E, Wood B et al (2012b) Blood cell origin of circulating microRNAs: a cautionary note for cancer biomarker studies. Cancer Prev Res 5:492–497CrossRefGoogle Scholar
  54. Redova M, Poprach A, Nekvindova J et al (2012) Circulating miR-378 and miR-451 in serum are potential biomarkers for renal cell carcinoma. J Transl Med 10:55CrossRefPubMedPubMedCentralGoogle Scholar
  55. Rottiers V, Naar AM (2012) MicroRNAs in metabolism and metabolic disorders. Nat Rev Mol Cell Biol 13:239–250CrossRefPubMedPubMedCentralGoogle Scholar
  56. Sanders I, Holdenrieder S, Walgenbach-Brünagel G et al (2012) Evaluation of reference genes for the analysis of serum miRNA in patients with prostate cancer, bladder cancer and renal cell carcinoma. Int J Urol 19:1017–1025CrossRefPubMedGoogle Scholar
  57. Schopman NC, Heynen S, Haasnoot J et al (2010) A miRNA-tRNA mix-up: tRNA origin of proposed miRNA. RNA Biol 7:573–576CrossRefPubMedGoogle Scholar
  58. Shende VR, Goldrick MM, Ramani S et al (2011) Expression and rhythmic modulation of circulating microRNAs targeting the clock gene Bmal1 in mice. PLoS ONE 6, e22586CrossRefPubMedPubMedCentralGoogle Scholar
  59. Silva J, García V, Zaballos Á et al (2010) Vesicle-related microRNAs in plasma of nonsmall cell lung cancer patients and correlation with survival. Eur Respir J 37:617–623CrossRefPubMedGoogle Scholar
  60. Singh P, Soon PS, Feige JJ et al (2011) Dysregulation of microRNAs in adrenocortical tumors. Mol Cell Endocrinol 351:118–128CrossRefPubMedGoogle Scholar
  61. Sivapragasam M, Rotondo F, Lloyd RV et al (2011) MicroRNAs in the human pituitary. Endocr Pathol 22:134–143CrossRefPubMedGoogle Scholar
  62. Song J, Bai Z, Han W et al (2011) Identification of suitable reference genes for qPCR analysis of serum microRNA in gastric cancer patients. Dig Dis Sci 57:897–904CrossRefPubMedGoogle Scholar
  63. Szabó PM, Butz H, Igaz P et al (2013) Minireview: miRomics in endocrinology: a novel approach for modeling endocrine diseases. Mol Endocrinol 274:573–585CrossRefGoogle Scholar
  64. Turchinovich A, Weiz L, Langheinz A et al (2011) Characterization of extracellular circulating microRNA. Nucleic Acids Res 39:7223–7233CrossRefPubMedPubMedCentralGoogle Scholar
  65. Turchinovich A, Weiz L, Burwinkel B (2012) Extracellular miRNAs: the mystery of their origin and function. Trends Biochem Sci 37:460–465CrossRefPubMedGoogle Scholar
  66. Van Ness J, Van Ness LK, Galas DJ (2003) Isothermal reactions for the amplification of oligonucleotides. Proc Natl Acad Sci USA 100:4504–4509CrossRefPubMedPubMedCentralGoogle Scholar
  67. Vlassov AV, Magdaleno S, Setterquist R et al (2012) Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta 1820:940–948CrossRefPubMedGoogle Scholar
  68. von Brandenstein M, Pandarakalam JJ, Kroon L et al (2012) MicroRNA 15a, inversely correlated to PKCα, is a potential marker to differentiate between benign and malignant renal tumors in biopsy and urine samples. Am J Pathol 180:1787–1797CrossRefGoogle Scholar
  69. Wang B, Howel P, Bruheim S et al (2011) Systematic evaluation of three microRNA profiling platforms: microarray, beads array, and quantitative real-time PCR array. PLoS ONE 6, e17167CrossRefPubMedPubMedCentralGoogle Scholar
  70. Wang K, Yuan Y, Cho JH et al (2012) Comparing the MicroRNA spectrum between serum and plasma. PLoS ONE 7, e41561CrossRefPubMedPubMedCentralGoogle Scholar
  71. Witwer KW (2015) Circulating microRNA biomarker studies: pitfalls and potential solutions. Clin Chem 61:56–63CrossRefPubMedGoogle Scholar
  72. Witwer KW, Buzás EI, Bemis LT et al (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2.doi: 10.3402/jev.v2i0.20360
  73. Yamada A, Cox MA, Gaffney KA et al (2014) Technical factors involved in the measurement of circulating microRNA biomarkers for the detection of colorectal neoplasia. PLoS ONE 9, e112481CrossRefPubMedPubMedCentralGoogle Scholar
  74. Zen K, Zhang CY (2012) Circulating microRNAs: a novel class of biomarkers to diagnose and monitor human cancers. Med Res Rev 32:326–348CrossRefPubMedGoogle Scholar
  75. Zhang J, Guo H, Qian G (2010) MiR-145, a new regulator of the DNA fragmentation factor-45 (DFF45)-mediated apoptotic network. Mol Cancer 9:211CrossRefPubMedPubMedCentralGoogle Scholar
  76. Zhang J, Zhao H, Gao Y et al (2012) Secretory miRNAs as novel cancer biomarkers. Biochim Biophys Acta 1826:32–43PubMedGoogle Scholar
  77. Zheng G, Wang H, Zhang X et al (2013) Identification and validation of reference genes for qPCR detection of serum microRNAs in colorectal adenocarcinoma patients. PLoS ONE 8, e83025CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  1. 1.Molecular Medicine Research GroupHungarian Academy of Sciences and Semmelweis UniversityBudapestHungary
  2. 2.Hungarian Academy of Sciences and Semmelweis University “Lendület” Hereditary Endocrine Tumors Research GroupBudapestHungary
  3. 3.Department of Laboratory MedicineSemmelweis UniversityBudapestHungary

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