Expression profiling of circulating miRNAs as a novel non-invasive diagnostic tool

Posted: 13 December 2011 |

Cell-free nucleic acids circulating in human blood were first described in 19481. However, it was not until the work of Sorengon and colleagues was published in 19942 that the importance of circulating nucleic acid (cfNA) was recognised. Today, the detection of diverse type of cfNA3 in blood and other body fluids is a valuable resource for the identification of a novel biomarker4,5. Although different types of cfNA have been described (including DNA, mRNA and microRNA), this review focuses on the isolation, detection and clinical utility of circulating microRNAs.

microRNAs (miRNAs) are an abundant class of short single stranded non-coding RNAs (~22 nts) that regulate gene expression at the posttranscriptional level. Interaction between an miRNA and any given of its mRNA targets results in either translation inhibition, mRNA degradation or a combination of both mechanisms. Therefore, miRNAs activity effectively reduces the transcriptional output of a target gene, without affecting its transcription rate. Currently, the sequence of over 60,000 microRNAs are deposited in the miRBase database [Version 17, April 20116]. miRNA activity has been associated with the control of a wide range of basic processes such as development, differentiation and metabolism. Detection of differential expression of miRNAs in many cases have established the basis for miRNA functional analysis and specific miRNA expression patterns can provide valuable diagnostic and prognostic indications, for example, in the context of human malignancies7,8. Moreover, the deregulation of the expression of miRNAs has been shown to contribute to cancer development through various kinds of mechanisms, including deletions, amplification or mutations involving miRNA loci, epigenetic silencing, as well as the dysregulation of transcription factors that target specific miRNAs9,10.

Circulating miRNA; a novel non-invasive diagnostic tool

Recently, cell-free miRNA have been isolated and measured in the blood11. The mechanism of how cell-free miRNAs are released into the circulation is yet unclear. To date, two different populations of circulating miRNAs have been identified; one associated to vesicles (exosomes and apoptotic bodies) and one associated to the Argonaute protein 2 [Ago212]. Although cellular fragmentation resulting from apoptotic processes is thought to be at the origin of circulating miRNA, increasing evidence suggests that not only exosomes-associated miRNAs can be actively secreted9, but also that miRNA-containing microvesicles can be efficiently delivered into recipient cells13,14. However, whether the levels of circulating miRNAs are able to regulate or coordinate gene expression at endocrine or paracrine level15 it is a crucial question that needs to be elucidated. The existence of a specific population of circulating miRNAs in the blood of healthy as well as diseased individuals have raised the possibility that changes in miRNA levels may serve as a biomarker for diagnosis, prognosis and therapeutic response of patients. Importantly, these miRNAs are stable, even after multiple freeze-thaw cycles16, which is an important prerequisite for utility as a biomarker17 and can be easily detected by using microarray18 or quantitative real-time PCR [qPCR19], and even Next Generation Sequencing [NGS20] approaches.

Purification and quantification of circulating miRNAs

During the past few years, our lab has developed and optimised dedicated protocols for the quantification and analysis of miRNA expression profiling in tissues and cells by using microarray21 and qPCR (miQPCR, manuscript in preparation). Furthermore, we have recently shown that the reliability and reproducibility of miRNA profiling are a function of the quality of the RNA used as input material22. Thus, a robust method for RNA isolation is essential. Because of the great interest in detecting circulating miRNAs, new dedicated protocols and products are emerging throughout the literature and in the portfolio of biotech companies (i.e. Exiqon). Technically, there are three major challenges associated to the analysis of circulating miRNAs: i) plasma and serum contain polymerase inhibitors; ii) isolated nucleic acids are too diluted to be easily detected; and iii) no endogenous reference genes are available for normalisation23. Technical approaches have been developed to solve problems associated to point (i) and (ii). However, the biggest challenge is the lack of reliable reference genes. As a matter of fact, qPCR data of miRNA levels in plasma of patients affected from the same type of tumour but carried out in different studies show conflicting results24. The observed discrepancies are most likely due to the lack of common reference genes. Suggested solutions include using a microarray-like approach by performing a median normalisation of the complete qPCR data25. However, this type of normalisation is feasible only if a large enough number of miRNAs and samples are analysed, leaving small scale projects with the problem of selecting and evaluating universal reference genes. As an alternative, we have developed an approach where carrier RNA, containing yeast RNA and artificial miRNAs, is added to the samples before RNA purification. This approach enables us to estimate the amount of RNA which is recovered (or lost) during RNA extraction. In addition, the employment of the spike-ins controls (i.e. artificial miRNAs) as reference genes allows the assessment and correction for differences in RNA recovery, cDNA synthesis as well as qPCR efficiency due to the presence of polymerase inhibitors.

Analysis of circulating microRNAs by using quantitative real-time PCR

Recently, our group has developed a novel approach to synthesise cDNA that we have named miQPCR (manuscript in preparation). The miQPCR method uniformly elongates all miRNAs contained in the sample by supplying a universal sequence. The extended miRNAs are then converted to cDNA through standard reverse transcription protocols. The advantage of the miQPCR over existing universal elongation approaches (i.e. polyadenylation) is that the addition of the linker extends all the miRNAs within the sample of a specific sequence. This allows both an increased efficiency in cDNA synthesis, as well as enabling flexible miRNA Specific Primers (miSPs) design. In fact, our innovative approach allows the design of temperature adjusted miSP to such an extent that virtually all primers we design have a predicted temperature of 55-60°C (which is the optimal temperature range for qPCR). Importantly, this is achieved without the necessity of modified nucleotide [i.e., LNA26 or ZNA27]. Our current work is focused on adapting the miQPCR approach to suit the detection of circulating miRNA in serum and plasma prepared from human and mouse blood.

Circulating miRNAs are potential biomarker for the detection of human diseases

During the past decade, miRNA activity has been associated with the control of a wide range of processes such as development, differentiation and metabolism28. Furthermore, it has been shown that dysfunctional expression of miRNAs is associated to the development of a number of human diseases, including cancer9,10. Several studies have identified miRNA expression signatures that classify cancer patients into different prognostic groups and survival prediction7,8. However, this experimental approach requires the isolation of cancer material through invasive techniques, limiting the effective diagnosis and surveillance of these complex multi-factorial diseases.

With this premise, circulating miRNAs have been ‘tagged’ by the biomedical community as easily accessible biomarkers for the screening of cancer patients. Indeed, during the past two years, several studies have been published. For example, the levels of circulating miRNAs have been reported to be potential diagnostic and prognostic biomarkers for different types of cancer including but not limited to breast cancer19,29, lung cancer20,30, prostate cancer31 and hepatocellular carcinoma32,33. In addition, Brase and colleagues31 have identified that levels of circulating mir-375 and miR-141 are not only able to positively identify prostate cancer, but also that their relative amount correlated with clinicpathological variables of tumour progression. Furthermore, the utility of circulating miRNAs as biomarkers is not limited to cancer, and although cancer studies are the ones making the headlines, different types of diseases can be assessed by using this methodological approach. In fact, the analysis of circulating miRNAs has been described as informative in the classification of patients with cardiovascular diseases25,34, coronary artery disease23, and chronic kidney disease. Furthermore, analysis of circulating miRNAs as biomarkers has shown great potential in the analysis and stratification of patients with different type of liver disease, including non alcoholic fatty liver disease [NAFLD36] alcoholic liver disease [ALD37] as well as and viral hepatitis type B and C36-38.


One of the major challenges towards an improved treatment of human diseases is the identification of appropriate markers for an early detection of diseases as well as for monitoring disease progression and patients’ response to therapy. In this review, we discussed and highlighted the potential clinical utility of circulating miRNAs as blood biomarkers. Clearly the profiling of circulating miRNAs is still in its infancy; additionally, the development of dedicated methodological approaches has just begun. However, the wealth of information that has been already published strongly suggests that circulating miRNAs will maintain their promise as biomarkers and that this fields it is here to stay and to thrive. Furthermore, it easy to predict that not only the profiling of circulating miRNAs will find its way ‘away’ from the bench into the clinical and diagnostic environments, but that it will also find novel application such as monitoring the efficacy of anticancer or anti-viral therapies, as well as having the potential of becoming themselves (i.e. the circulating miRNAs) a future therapeutic targets for the treatment of human diseases.

About the Author

Mirco Castoldi is a molecular and cellular biologist that during his studies has explored different fields of basic research. Since achieving his PhD in 2004, he has joined the field of Molecular Medicine, where he investigated microRNA activity in the biogenesis of human disease. His fundamental contribution has lead to the development of microRNA expression profiling platform by using microarray (miCHIP) and qPCR (miQPCR). Recently, Mirco has discovered that the liver specific microRNA miR-122 has the potential to regulate systemic iron homeostasis. His current research focuses on elucidating the role played by microRNAs in controlling systemic and cellular iron homeostasis in health and disease.


1. Mandel, P.M., P. (1948) Les acides nucléiques du plasma sanguin chez l’homme. C. R. Acad. Sci. Paris, 142, 241–243

2. Sorenson, G.D., Pribish, D.M., Valone, F.H., Memoli, V.A., Bzik, D.J. and Yao, S.L. (1994) Soluble normal and mutated DNA sequences from single-copy genes in human blood. Cancer Epidemiol Biomarkers Prev, 3, 67-71

3. Schwarzenbach, H., Hoon, D.S. and Pantel, K. (2011) Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer, 11, 426-437

4. Brase, J.C., Wuttig, D., Kuner, R. and Sultmann, H. (2010) Serum microRNAs as non-invasive biomarkers for cancer. Mol Cancer, 9, 306

5. Tzimagiorgis, G., Michailidou, E.Z., Kritis, A., Markopoulos, A.K. and Kouidou, S. (2011) Recovering circulating extracellular or cell-free RNA from bodily fluids. Cancer Epidemiol

6. Griffiths-Jones, S., Saini, H.K., van Dongen, S. and Enright, A.J. (2008) miRBase: tools for microRNA genomics. Nucleic Acids Res, 36, D154-158

7. Li, X., Zhang, Y., Ding, J., Wu, K. and Fan, D. (2010) Survival prediction of gastric cancer by a sevenmicroRNA signature. Gut, 59, 579-585

8. Srinivasan, S., Patric, I.R. and Somasundaram, K. (2011) A ten-microRNA expression signature predicts survival in glioblastoma. PLoS One, 6, e17438

9. Yendamuri, S. and Calin, G.A. (2009) The role of microRNA in human leukemia: a review. Leukemia, 23, 1257-1263

10. Ladeiro, Y., Couchy, G., Balabaud, C., Bioulac-Sage, P., Pelletier, L., Rebouissou, S. and Zucman-Rossi, J. (2008) MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/ tumor suppressor gene mutations. Hepatology, 47, 1955-1963

11. Chim, S.S., Shing, T.K., Hung, E.C., Leung, T.Y., Lau, T.K., Chiu, R.W. and Lo, Y.M. (2008) Detection and characterization of placental microRNAs in maternal plasma. Clin Chem, 54, 482-490

12. Arroyo, J.D., Chevillet, J.R., Kroh, E.M., Ruf, I.K., Pritchard, C.C., Gibson, D.F., Mitchell, P.S., Bennett, C.F., Pogosova- Agadjanyan, E.L., Stirewalt, D.L. et al. (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A, 108, 5003-5008

13. Skog, J., Wurdinger, T., van Rijn, S., Meijer, D.H., Gainche, L., Sena-Esteves, M., Curry, W.T., Jr., Carter, B.S., Krichevsky, A.M. and Breakefield, X.O. (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol, 10, 1470-1476

14. Deregibus, M.C., Cantaluppi, V., Calogero, R., Lo Iacono, M., Tetta, C., Biancone, L., Bruno, S., Bussolati, B. and Camussi, G. (2007) Endothelial progenitor cell derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA. Blood, 110, 2440-2448

15. Gupta, S.K., Bang, C. and Thum, T. (2010) Circulating microRNAs as biomarkers and potential paracrine mediators of cardiovascular disease. Circ Cardiovasc Genet, 3, 484-488

16. Gilad, S., Meiri, E., Yogev, Y., Benjamin, S., Lebanony, D., Yerushalmi, N., Benjamin, H., Kushnir, M., Cholakh, H., Melamed, N. et al. (2008) Serum microRNAs are promising novel biomarkers. PLoS One, 3, e3148

17. Mitchell, P.S., Parkin, R.K., Kroh, E.M., Fritz, B.R., Wyman, S.K., Pogosova-Agadjanyan, E.L., Peterson, A., Noteboom, J., O’Briant, K.C., Allen, A. et al. (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A, 105, 10513-10518

18. Keller, A., Leidinger, P., Bauer, A., ElSharawy, A., Haas, J., Backes, C., Wendschlag, A., Giese, N., Tjaden, C., Ott, K. et al. (2011) Toward the blood-borne miRNome of human diseases. Nature Methods, 8, 841-U104

19. Zhao, H., Shen, J., Medico, L., Wang, D., Ambrosone, C.B. and Liu, S. (2010) A pilot study of circulating miRNAs as potential biomarkers of early stage breast cancer. PLoS One, 5, e13735

20. Keller, A., Backes, C., Leidinger, P., Kefer, N., Boisguerin, V., Barbacioru, C., Vogel, B., Matzas, M., Huwer, H., Katus, H.A. et al. (2011) Next-generation sequencing identifies novel microRNAs in peripheral blood of lung cancer patients. Mol Biosyst

21. Castoldi, M., Schmidt, S., Benes, V., Noerholm, M., Kulozik, A.E., Hentze, M.W. and Muckenthaler, M.U. (2006) A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA). Rna, 12, 913-920

22. Ibberson, D., Benes, V., Muckenthaler, M.U. and Castoldi, M. (2009) RNA degradation compromises the reliability of microRNA expression profiling. BMC Biotechnol, 9, 102

23. Fichtlscherer, S., De Rosa, S., Fox, H., Schwietz, T., Fischer, A., Liebetrau, C., Weber, M., Hamm, C.W., Roxe, T., Muller- Ardogan, M. et al. (2010) Circulating microRNAs in patients with coronary artery disease. Circ Res, 107, 677-684

24. Kosaka, N., Iguchi, H. and Ochiya, T. (2010) Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis. Cancer Sci, 101, 2087-2092

25. Mestdagh, P., Van Vlierberghe, P., De Weer, A., Muth, D., Westermann, F., Speleman, F. and Vandesompele, J. (2009) A novel and universal method for microRNA RTqPCR data normalization. Genome Biol, 10, R64

26. Hojland, T., Kumar, S., Babu, B.R., Umemoto, T., Albaek, N., Sharma, P.K., Nielsen, P. and Wengel, J. (2007) LNA (locked nucleic acid) and analogs as triplex-forming oligonucleotides. Org Biomol Chem, 5, 2375-2379

27. Moreau, V., Voirin, E., Paris, C., Kotera, M., Nothisen, M., Remy, J.S., Behr, J.P., Erbacher, P. and Lenne-Samuel, N. (2009) Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription. Nucleic Acids Res, 37, e130

28. Krutzfeldt, J. and Stoffel, M. (2006) MicroRNAs: a new class of regulatory genes affecting metabolism. Cell Metab, 4, 9-12

29. Ross, J.S. (2010) Measuring circulating miRNAs: the new “PSA” for Breast Cancer? Oncologist, 15, 656

30. Heneghan, H.M., Miller, N. and Kerin, M.J. (2010) Circulating miRNA signatures: promising prognostic tools for cancer. J Clin Oncol, 28, e573-574; author reply e575-576

31. Brase, J.C., Johannes, M., Schlomm, T., Falth, M., Haese, A., Steuber, T., Beissbarth, T., Kuner, R. and Sultmann, H. (2011) Circulating miRNAs are correlated with tumor progression in prostate cancer. Int J Cancer, 128, 608-616

32. Qu, K.Z., Zhang, K., Li, H., Afdhal, N.H. and Albitar, M. (2011) Circulating microRNAs as biomarkers for hepatocellular carcinoma. J Clin Gastroenterol, 45, 355-360

33. Tomimaru, Y., Eguchi, H., Nagano, H., Wada, H., Kobayashi, S., Marubashi, S., Tanemura, M., Tomokuni, A., Takemasa, I., Umeshita, K. et al. (2011) Circulating microRNA-21 as a novel biomarker for hepatocellular carcinoma. J Hepatol

34. Dimmeler, S. and Zeiher, A.M. (2010) Circulating microRNAs: novel biomarkers for cardiovascular diseases? Eur Heart J, 31, 2705-2707

35. Neal, C.S., Michael, M.Z., Pimlott, L.K., Yong, T.Y., Li, J.Y. and Gleadle, J.M. (2011) Circulating microRNA expression is reduced in chronic kidney disease. Nephrol Dial Transplant

36. Cermelli, S., Ruggieri, A., Marrero, J.A., Ioannou, G.N. and Beretta, L. (2011) Circulating MicroRNAs in Patients with Chronic Hepatitis C and Non-Alcoholic Fatty Liver Disease. PLoS One, 6, e23937

37. Zhang, Y., Jia, Y., Zheng, R., Guo, Y., Wang, Y., Guo, H., Fei, M. and Sun, S. (2010) Plasma microRNA-122 as a biomarker for viral-, alcohol-, and chemical-related hepatic diseases. Clin Chem, 56, 1830-1838

38. Ji, F., Yang, B., Peng, X., Ding, H., You, H. and Tien, P. (2011) Circulating microRNAs in hepatitis B virus-infected patients. J Viral Hepat, 18, e242-251

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