Circulating microRNA biomarkers as liquid biopsy for cancer patients: Pros and cons of current assays

Journal of Clinical Medicine

Volumn 4, Issue 10, 2015, Pages 1890-1907

  Ono, Shigeshi ^a   Lam, Stella ^a   Nagahara, Makoto ^a   Hoon, Dave S B ^a  

^a JOHN WAYNE CANCER INSTITUTE   (United States)

Author keywords

Blood; Cancer patients; Circulating microRNA; Circulating nucleic acids; Diagnosis; Next generation sequencing; Prognosis

Indexed keywords

CIRCULATING MICRORNA; MICRORNA; MICRORNA 107; MICRORNA 142 30B; MICRORNA 142 3P; MICRORNA 16; MICRORNA 21; MICRORNA 210; MICRORNA 238; MICRORNA 29B; MICRORNA 39; MICRORNA 422B; MICRORNA 54; UNCLASSIFIED DRUG;

BREAST CANCER; CANCER DIAGNOSIS; CANCER PATIENT; CANCER PROGNOSIS; CANCER STAGING; CAPILLARY ELECTROPHORESIS; DISEASE FREE SURVIVAL; HUMAN; MEASUREMENT ACCURACY; MICROARRAY ANALYSIS; NEXT GENERATION SEQUENCING; NONHUMAN; REPRODUCIBILITY; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; REVIEW; RNA EXTRACTION; SENSITIVITY AND SPECIFICITY; TUMOR BIOPSY; TUMOR GROWTH;

EID: 84959093704     PISSN: None     EISSN: 20770383     Source Type: Journal    
DOI: 10.3390/jcm4101890     Document Type: Review

References (82)

1. Kim, V.N. Microrna biogenesis: Coordinated cropping and dicing. Nat. Rev. Mol. Cell Biol. 2005, 6, 376–385.
2. Ambros, V. Micrornas: Tiny regulators with great potential. Cell 2001, 107, 823–826.
3. Bartel, D.P. Micrornas: Genomics, biogenesis, mechanism, and function. Cell 2004, 116, 281–297.
4. Reinhart, B.J., Slack, F.J., Basson, M., Pasquinelli, A.E., Bettinger, J.C., Rougvie, A.E., Horvitz, H.R., Ruvkun, G. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 2000, 403, 901–906.
5. Krek, A., Grun, D., Poy, M.N., Wolf, R., Rosenberg, L., Epstein, E.J., MacMenamin, P., da Piedade, I., Gunsalus, K.C., Stoffel, M., et al. Combinatorial microRNA target predictions. Nat. Genet. 2005, 37, 495–500.
6. Filipowicz, W., Bhattacharyya, S.N., Sonenberg, N. Mechanisms of post-transcriptional regulation by microRNAs: Are the answers in sight? Nat. Rev. Genet. 2008, 9, 102–114.
7. Calin, G.A., Dumitru, C.D., Shimizu, M., Bichi, R., Zupo, S., Noch, E., Aldler, H., Rattan, S., Keating, M., Rai, K., et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA 2002, 99, 15524–15529.
8. Takamizawa, J., Konishi, H., Yanagisawa, K., Tomida, S., Osada, H., Endoh, H., Harano, T., Yatabe, Y., Nagino, M., Nimura, Y., et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res. 2004, 64, 3753–3756.
9. Calin, G.A., Ferracin, M., Cimmino, A., Di Leva, G., Shimizu, M., Wojcik, S.E., Iorio, M.V., Visone, R., Sever, N.I., Fabbri, M., et al. A microRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N. Engl. J. Med. 2005, 353, 1793–1801.
10. Croce, C.M. Causes and consequences of microRNA dysregulation in cancer. Nat. Rev. Genet. 2009, 10, 704–714.
11. Kasinski, A.L., Slack, F.J. Epigenetics and genetics. MicroRNAs en route to the clinic: Progress in validating and targeting microRNAs for cancer therapy. Nat. Rev. Cancer 2011, 11, 849–864.
12. Marzese, D.M., Hirose, H., Hoon, D.S. Diagnostic and prognostic value of circulating tumor-related DNA in cancer patients. Expert Rev. Mol. Diagn. 2013, 13, 827–844.
13. Crowley, E., Di Nicolantonio, F., Loupakis, F., Bardelli, A. Liquid biopsy: Monitoring cancer-genetics in the blood. Nat. Rev. Clin. Oncol. 2013, 10, 472–484.
14. Clinical Laboratory Improvement Amendments (CLIA). Available online: http://www.fda.gov/medicaldevices/deviceregulationandguidance/ivdregulatoryassistance/ucm124105.htm (accessed on 1 August 2015).
15. Hoshimoto, S., Shingai, T., Morton, D.L., Kuo, C., Faries, M.B., Chong, K., Elashoff, D., Wang, H.J., Elashoff, R.M., Hoon, D.S. Association between circulating tumor cells and prognosis in patients with stage ш melanoma with sentinel lymph node metastasis in a phase ш international multicenter trial. J. Clin. Oncol. 2012, 30, 3819–3826.
16. Weber, J.A., Baxter, D.H., Zhang, S., Huang, D.Y., Huang, K.H., Lee, M.J., Galas, D.J., Wang, K. The microRNA spectrum in 12 body fluids. Clin. Chem. 2010, 56, 1733–1741.
17. Watson, A.K., Witwer, K.W. Do platform-specific factors explain microRNA profiling disparities? Clin. Chem. 2012, 58, 472–474; author reply 474–475.
18. 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. Circulating microRNAs as stable blood-based markers for cancer detection. Proc. Natl. Acad. Sci. USA 2008, 105, 10513–10518.
19. Schwarzenbach, H., Hoon, D.S., Pantel, K. Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer 2011, 11, 426–437.
20. Sourvinou, I.S., Markou, A., Lianidou, E.S. Quantification of circulating mirRNA in plasma: Effect of preanalytical and analytical parameters on their isolation and stability. J. Mol. Diagn. 2013, 15, 827–834.
21. Montani, F., Marzi, M.J., Dezi, F., Dama, E., Carletti, R.M., Bonizzi, G., Bertolotti, R., Bellomi, M., Rampinelli, C., Maisonneuve, P., et al. MiR-test: A blood test for lung cancer early detection. J. Natl. Cancer Inst. 2015, 107 djv063, doi:10.1093/jnci/djv063.
22. Pritchard, C.C., Cheng, H.H., Tewari, M. MicroRNA profiling: Approaches and considerations. Nat. Rev. Genet. 2012, 13, 358–369.
23. Kirschner, M.B., van Zandwijk, N., Reid, G. Cell-free microRNAs: Potential biomarkers in need of standardized reporting. Front. Genet. 2013, 4, 56, doi:10.3389/fgene.2013.00056.
24. Lawrie, C.H., Gal, S., Dunlop, H.M., Pushkaran, B., Liggins, A.P., Pulford, K., Banham, A.H., Pezzella, F., Boultwood, J., Wainscoat, J.S., et al. Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large b-cell lymphoma. Br. J. Haematol. 2008, 141, 672–675.
25. Heegaard, N.H., Schetter, A.J., Welsh, J.A., Yoneda, M., Bowman, E.D., Harris, C.C. Circulating micro-RNA expression profiles in early stage nonsmall cell lung cancer. Int. J. Cancer 2012, 130, 1378–1386.
26. Hennessey, P.T., Sanford, T., Choudhary, A., Mydlarz, W.W., Brown, D., Adai, A.T., Ochs, M.F., Ahrendt, S.A., Mambo, E., Califano, J.A. Serum microRNA biomarkers for detection of non-small cell lung cancer. PLoS ONE 2012, 7, e32307, doi:10.1371/journal.pone.0032307.
27. Hu, Z., Chen, X., Zhao, Y., Tian, T., Jin, G., Shu, Y., Chen, Y., Xu, L., Zen, K., Zhang, C., et al. Serum microRNA signatures identified in a genome-wide serum microRNA expression profiling predict survival of non-small-cell lung cancer. J. Clin. Oncol. 2010, 28, 1721–1726.
28. Silva, J., Garcia, V., Zaballos, A., Provencio, M., Lombardia, L., Almonacid, L., Garcia, J.M., Dominguez, G., Pena, C., Diaz, R., et al. Vesicle-related microRNAs in plasma of nonsmall cell lung cancer patients and correlation with survival. Eur. Respir. J. 2011, 37, 617–623.
29. Shen, J., Liu, Z., Todd, N.W., Zhang, H., Liao, J., Yu, L., Guarnera, M.A., Li, R., Cai, L., Zhan, M., et al. Diagnosis of lung cancer in individuals with solitary pulmonary nodules by plasma microRNA biomarkers. BMC Cancer 2011, 11, 374, doi:10.1186/1471-2407-11-374.
30. Shen, J., Wang, A., Wang, Q., Gurvich, I., Siegel, A.B., Remotti, H., Santella, R.M. Exploration of genome-wide circulating microRNA in hepatocellular carcinoma: MiR-483–5p as a potential biomarker. Cancer Epidemiol. Biomark. Prev. 2013, 22, 2364–2373.
31. Summerer, I., Niyazi, M., Unger, K., Pitea, A., Zangen, V., Hess, J., Atkinson, M.J., Belka, C., Moertl, S., Zitzelsberger, H. Changes in circulating microRNAs after radiochemotherapy in head and neck cancer patients. Radiat. Oncol. 2013, 8, 296, doi:10.1186/1748-717X-8-296.
32. Zhu, C., Ren, C., Han, J., Ding, Y., Du, J., Dai, N., Dai, J., Ma, H., Hu, Z., Shen, H., et al. A five-microRNA panel in plasma was identified as potential biomarker for early detection of gastric cancer. Br. J. Cancer 2014, 110, 2291–2299.
33. Zhou, J., Yu, L., Gao, X., Hu, J., Wang, J., Dai, Z., Wang, J.F., Zhang, Z., Lu, S., Huang, X., et al. Plasma microRNA panel to diagnose hepatitis b virus-related hepatocellular carcinoma. J. Clin. Oncol. 2011, 29, 4781–4788.
34. Wulfken, L.M., Moritz, R., Ohlmann, C., Holdenrieder, S., Jung, V., Becker, F., Herrmann, E., Walgenbach-Brunagel, G., von Ruecker, A., Muller, S.C., et al. MicroRNAs in renal cell carcinoma: Diagnostic implications of serum mir-1233 levels. PLoS ONE 2011, 6, e25787, doi:10.1371/journal.pone.0025787.
35. Asaga, S., Kuo, C., Nguyen, T., Terpenning, M., Giuliano, A.E., Hoon, D.S. Direct serum assay for microRNA-21 concentrations in early and advanced breast cancer. Clin. Chem. 2011, 57, 84–91.
36. Ono, S., Oyama, T., Lam, S., Chong, K., Foshag, L.J., Hoon, D.S. A direct plasma assay of circulating microRNA-210 of hypoxia can identify early systemic metastasis recurrence in melanoma patients. Oncotarget 2015, 6, 7053–7064.
37. Rocci, A., Hofmeister, C.C., Geyer, S., Stiff, A., Gambella, M., Cascione, L., Guan, J., Benson, D.M., Efebera, Y.A., Talabere, T., et al. Circulating mirna markers show promise as new prognosticators for multiple myeloma. Leukemia 2014, 28, 1922–1926.
38. Witwer, K.W., Buzas, E.I., Bemis, L.T., Bora, A., Lasser, C., Lotvall, J., Nolte-’t Hoen, E.N., Piper, M.G., Sivaraman, S., Skog, J., et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J. Extracell. Vesicles 2013, 2, doi:10.3402/jev.v2i0.20360.
39. Kannan, M., Atreya, C. Differential profiling of human red blood cells during storage for 52 selected microrRNA. Transfusion 2010, 50, 1581–1588.
40. Chen, X., Ba, Y., Ma, L., Cai, X., Yin, Y., Wang, K., Guo, J., Zhang, Y., Chen, J., Guo, X., et al. Characterization of microRNAs in serum: A novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 2008, 18, 997–1006.
41. D’Alessandra, Y., Devanna, P., Limana, F., Straino, S., Di Carlo, A., Brambilla, P.G., Rubino, M., Carena, M.C., Spazzafumo, L., de Simone, M., et al. Circulating microRNAs are new and sensitive biomarkers of myocardial infarction. Eur. Heart J. 2010, 31, 2765–2773.
42. Wang, K., Yuan, Y., Cho, J.H., McClarty, S., Baxter, D., Galas, D.J. Comparing the microRNA spectrum between serum and plasma. PLoS ONE 2012, 7, e41561, doi:10.137/journal.pone.0041561.
43. McDonald, J.S., Milosevic, D., Reddi, H.V., Grebe, S.K., Algeciras-Schimnich, A. Analysis of circulating microRNA: Preanalytical and analytical challenges. Clin. Chem. 2011, 57, 833–840.
44. Cheng, H.H., Yi, H.S., Kim, Y., Kroh, E.M., Chien, J.W., Eaton, K.D., Goodman, M.T., Tait, J.F., Tewari, M., Pritchard, C.C. Plasma processing conditions substantially influence circulating microRNA biomarker levels. PLoS ONE 2013, 8, e64795, doi:10.1371/journal.pone.0064795.
45. Hunter, M.P., Ismail, N., Zhang, X., Aguda, B.D., Lee, E.J., Yu, L., Xiao, T., Schafer, J., Lee, M.L., Schmittgen, T.D., et al. Detection of microRNA expression in human peripheral blood microvesicles. PLoS ONE 2008, 3, e3694, doi:10.1371/journal.pone.0003694.
46. Willeit, P., Zampetaki, A., Dudek, K., Kaudewitz, D., King, A., Kirkby, N.S., Crosby-Nwaobi, R., Prokopi, M., Drozdov, I., Langley, S.R., et al. Circulating microRNAs as novel biomarkers for platelet activation. Circ. Res. 2013, 112, 595–600.
47. Al-Soud, W.A., Radstrom, P. Purification and characterization of PCR-inhibitory components in blood cells. J. Clin. Microbiol. 2001, 39, 485–493.
48. Boeckel, J.N., Thome, C.E., Leistner, D., Zeiher, A.M., Fichtlscherer, S., Dimmeler, S. Heparin selectively affects the quantification of microRNAs in human blood samples. Clin. Chem. 2013, 59, 1125–1127.
49. Kaudewitz, D., Lee, R., Willeit, P., McGregor, R., Markus, H.S., Kiechl, S., Zampetaki, A., Storey, R.F., Channon, K.M., Mayr, M. Impact of intravenous heparin on quantification of circulating microRNAs in patients with coronary artery disease. Thromb. Haemost. 2013, 110, 609–615.
50. Kim, D.J., Linnstaedt, S., Palma, J., Park, J.C., Ntrivalas, E., Kwak-Kim, J.Y., Gilman-Sachs, A., Beaman, K., Hastings, M.L., Martin, J.N., et al. Plasma components affect accuracy of circulating cancer-related microRNA quantitation. J. Mol. Diagn. 2012, 14, 71–80.
51. Garcia, M.E., Blanco, J.L., Caballero, J., Gargallo-Viola, D. Anticoagulants interfere with PCR used to diagnose invasive aspergillosis. J. Clin. Microbiol. 2002, 40, 1567–1568.
52. 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. Circulating microRNAs in patients with coronary artery disease. Circ. Res. 2010, 107, 677–684.
53. Ma, W., Wang, M., Wang, Z.Q., Sun, L., Graber, D., Matthews, J., Champlin, R., Yi, Q., Orlowski, R.Z., Kwak, L.W., et al. Effect of long-term storage in TRIzol on microarray-based gene expression profiling. Cancer Epidemiol. Biomarkers Prev. 2010, 19, 2445–2452.
54. Kim, Y.K., Yeo, J., Kim, B., Ha, M., Kim, V.N. Short structured RNAs with low GC content are selectively lost during extraction from a small number of cells. Mol. Cell 2012, 46, 893–895.
55. Rio, D.C., Ares, M., Jr., Hannon, G.J., Nilsen, T.W. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb. Protoc. 2010, 2010, pdb.prot5439, doi:10.1101/pdb.prot5439.
56. Rio, D.C., Ares, M., Jr., Hannon, G.J., Nilsen, T.W. Guidelines for the use of RNA purification kits. Cold Spring Harb. Protoc. 2010, 2010, pdb.ip79, doi:10.1101/pdb.ip79.
57. Tosar, J.P., Gambaro, F., Sanguinetti, J., Bonilla, B., Witwer, K.W., Cayota, A. Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines. Nucleic Acids Res. 2015, 43, 5601–5616.
58. Witwer, K.W. Circulating microRNA biomarker studies: Pitfalls and potential solutions. Clin. Chem. 2015, 61, 56–63.
59. Kroh, E.M., Parkin, R.K., Mitchell, P.S., Tewari, M. Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcriptionPCR (qRT-PCR). Methods 2010, 50, 298–301.
60. Asaga, S., Hoon, D.S. Direct serum assay for microRNA in cancer patients. Methods Mol. Biol. 2013, 1024, 147–155.
61. Schroeder, A., Mueller, O., Stocker, S., Salowsky, R., Leiber, M., Gassmann, M., Lightfoot, S., Menzel, W., Granzow, M., Ragg, T. The rin: An RNA integrity number for assigning integrity values to RNA measurements. BMC Mol. Biol. 2006, 7, 3, doi:10.1186/1471-2199-7-3.
62. Jung, M., Schaefer, A., Steiner, I., Kempkensteffen, C., Stephan, C., Erbersdobler, A., Jung, K. Robust microRNA stability in degraded RNA preparations from human tissue and cell samples. Clin. Chem. 2010, 56, 998–1006.
63. Aqeilan, R.I., Calin, G.A., Croce, C.M. MiR-15a and miR-16–1 in cancer: Discovery, function and future perspectives. Cell Death Differ. 2010, 17, 215–220.
64. Wang, Y., Gao, Y., Shi, W., Zhai, D., Rao, Q., Jia, X., Liu, J., Jiao, X., Du, Z. Profiles of differential expression of circulating microrRNA in hepatitis b virus-positive small hepatocellular carcinoma. Cancer Biomark. 2015, 15, 171–180.
65. Filkova, M., Aradi, B., Senolt, L., Ospelt, C., Vettori, S., Mann, H., Filer, A., Raza, K., Buckley, C.D., Snow, M., et al. Association of circulating miR-223 and miR-16 with disease activity in patients with early rheumatoid arthritis. Ann. Rheum. Dis. 2014, 73, 1898–1904.
66. Haider, B.A., Baras, A.S., McCall, M.N., Hertel, J.A., Cornish, T.C., Halushka, M.K. A critical evaluation of microRNA biomarkers in non-neoplastic disease. PLoS ONE 2014, 9, e89565, doi:10.1371/journal.pone.0089565.
67. Xiang, M., Zeng, Y., Yang, R., Xu, H., Chen, Z., Zhong, J., Xie, H., Xu, Y., Zeng, X. U6 is not a suitable endogenous control for the quantification of circulating microrRNA. Biochem. Biophys. Res. Commun. 2014, 454, 210–214.
68. Zeng, X., Xiang, J., Wu, M., Xiong, W., Tang, H., Deng, M., Li, X., Liao, Q., Su, B., Luo, Z., et al. Circulating miR-17, miR-20a, miR-29c, and miR-223 combined as non-invasive biomarkers in nasopharyngeal carcinoma. PLoS ONE 2012, 7, e46367, doi:10.1371/journal.pone.0046367.
69. 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, 3983–3994.
70. Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., et al. The miqe guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009, 55, 611–622.
71. Huggett, J.F., Foy, C.A., Benes, V., Emslie, K., Garson, J.A., Haynes, R., Hellemans, J., Kubista, M., Mueller, R.D., Nolan, T., et al. The digital MIQE guidelines: Minimum information for publication of quantitative digital PCR experiments. Clin. Chem. 2013, 59, 892–902.
72. Li, N., Ma, J., Guarnera, M.A., Fang, H., Cai, L., Jiang, F. Digital PCR quantification of mirRNA in sputum for diagnosis of lung cancer. J. Cancer Res. Clin. Oncol. 2014, 140, 145–150.
73. Moldovan, L., Batte, K., Wang, Y., Wisler, J., Piper, M. Analyzing the circulating microRNAs in exosomes/extracellular vesicles from serum or plasma by qRT-PCR. Methods Mol. Biol. 2013, 1024, 129–145.
74. Git, A., Dvinge, H., Salmon-Divon, M., Osborne, M., Kutter, C., Hadfield, J., Bertone, P., Caldas, C. Systematic comparison of microarray profiling, real-time PCR, and next-generation sequencing technologies for measuring differential microrna expression. RNA 2010, 16, 991–1006.
75. Li, W., Ruan, K. MicroRNA detection by microarray. Anal. Bioanal. Chem. 2009, 394, 1117–1124.
76. Mestdagh, P., Hartmann, N., Baeriswyl, L., Andreasen, D., Bernard, N., Chen, C., Cheo, D., D’Andrade, P., DeMayo, M., Dennis, L., et al. Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study. Nat. Methods 2014, 11, 809–815.
77. Chen, Y., Gelfond, J.A., McManus, L.M., Shireman, P.K. Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis. BMC Genom. 2009, 10, 407, doi:10.1186/1471-2164-10-407.
78. Hansen, T.F., Carlsen, A.L., Heegaard, N.H., Sorensen, F.B., Jakobsen, A. Changes in circulating microRNA-126 during treatment with chemotherapy and bevacizumab predicts treatment response in patients with metastatic colorectal cancer. Br. J. Cancer 2015, 112, 624–629.
79. Komatsu, S., Ichikawa, D., Hirajima, S., Kawaguchi, T., Miyamae, M., Okajima, W., Ohashi, T., Arita, T., Konishi, H., Shiozaki, A., et al. Plasma microrna profiles: Identification of miR-25 as a novel diagnostic and monitoring biomarker in oesophageal squamous cell carcinoma. Br. J. Cancer 2014, 111, 1614–1624.
80. Tanaka, Y., Tsuda, S., Kunikata, H., Sato, J., Kokubun, T., Yasuda, M., Nishiguchi, K.M., Inada, T., Nakazawa, T. Profiles of extracellular mirnas in the aqueous humor of glaucoma patients assessed with a microarray system. Sci. Rep. 2014, 4, 5089, doi:10.1038/srep05089.
81. Ono, M., Kosaka, N., Tominaga, N., Yoshioka, Y., Takeshita, F., Takahashi, R.U., Yoshida, M., Tsuda, H., Tamura, K., Ochiya, T. Exosomes from bone marrow mesenchymal stem cells contain a microRNA that promotes dormancy in metastatic breast cancer cells. Sci. Signal. 2014, 7, 63, doi:10.1126/scisignal.2005231.
82. Thompson, D., Botros, I., Rounseville, M., Liu, Q., Wang, E., Harrison, H., Roche, P. Automated High Fidelity RNA Expression Profiling Using Nuclease Protection Coupled with next Generation Sequencing. Available online: http://www.htgmolecular.com/sites/default/files/Merck%20poster%2030-Apr-2014.pdf (accessed on 20 October 2015)