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Practical Procedures for Improving Detection of Circulating miRNAs in Cardiovascular Diseases

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Abstract

Hemolysis has been known to affect the measurement of circulating biomarkers. In this study, clinically applicable procedures for microRNA (miRNA) detection in serum samples of acute myocardial infarction patients were established. The 89 samples from patients admitted to the coronary care unit were collected. These samples obtained from heparin-treated and untreated patients were subjected to heparinase digestion prior to miRNA measurements by multiplex RT-qPCR. The good reproducibility of miRNA detection after heparinase digestion (average R2 = 0.97) indicated that this method can be used routinely for samples regardless of heparin medication. Additionally, the degree of hemolysis in these samples was highly related to the hemoglobin absorbance at 414 nm. Based on the hemoglobin absorbance, five hemolysis-associated miRNAs were identified in our data normalized with respect to both the spike-in control and the RNA amount in a given sample. Using these calibration procedures, miRNAs can be accurately quantified and identified for clinical samples.

The practical procedures for miRNA detection in serum samples from the coronary care unit were established, and five hemolysis-associated miRNAs were accurately clarified through serial normalization.

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Abbreviations

AMI:

acute myocardial infarction

CCU:

coronary care unit

conc.:

concentration

CV:

coefficient of variation

CVD:

cardiovascular disease

miRNA:

microRNA

RT-qPCR:

reverse transcription quantitative polymerase chain reaction

SD:

standard deviation

SE:

standard error

References

  1. Ha, M., & Kim, V. N. (2014). Regulation of microRNA biogenesis. Nature Reviews. Molecular Cell Biology, 15(8), 509–524. https://doi.org/10.1038/nrm3838.

    Article  CAS  PubMed  Google Scholar 

  2. Mendell, J. T., & Olson, E. N. (2012). MicroRNAs in stress signaling and human disease. Cell, 148(6), 1172–1187. https://doi.org/10.1016/j.cell.2012.02.005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Larrea, E., Sole, C., Manterola, L., Goicoechea, I., Armesto, M., Arestin, M., et al. (2016). New concepts in cancer biomarkers: circulating miRNAs in liquid biopsies. International Journal of Molecular Sciences, 17(5). https://doi.org/10.3390/ijms17050627.

  4. Fan, P. C., Chen, C. C., Chen, Y. C., Chang, Y. S., & Chu, P. H. (2016). MicroRNAs in acute kidney injury. Human Genomics, 10(1), 29. https://doi.org/10.1186/s40246-016-0085-z.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Barwari, T., Joshi, A., & Mayr, M. (2016). MicroRNAs in cardiovascular disease. Journal of the American College of Cardiology, 68(23), 2577–2584. https://doi.org/10.1016/j.jacc.2016.09.945.

    Article  CAS  PubMed  Google Scholar 

  6. Viereck, J., & Thum, T. (2017). Circulating noncoding RNAs as biomarkers of cardiovascular disease and injury. Circulation Research, 120(2), 381–399. https://doi.org/10.1161/CIRCRESAHA.116.308434.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  8. Kaudewitz, D., Lee, R., Willeit, P., McGregor, R., Markus, H. S., Kiechl, S., et al. (2013). Impact of intravenous heparin on quantification of circulating microRNAs in patients with coronary artery disease. Thrombosis and Haemostasis, 110(3), 609–615. https://doi.org/10.1160/TH13-05-0368.

    Article  CAS  PubMed  Google Scholar 

  9. Izraeli, S., Pfleiderer, C., & Lion, T. (1991). Detection of gene expression by PCR amplification of RNA derived from frozen heparinized whole blood. Nucleic Acids Research, 19(21), 6051. https://doi.org/10.1093/nar/19.21.6051.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Coelho-Lima, J., Mohammed, A., Cormack, S., Jones, S., Das, R., Egred, M., et al. (2018). Overcoming heparin-associated RT-qPCR inhibition and normalization issues for microRNA quantification in patients with acute myocardial infarction. Thrombosis and Haemostasis, 118(7), 1257–1269. https://doi.org/10.1055/s-0038-1660437.

    Article  PubMed  Google Scholar 

  11. Kondratov, K., Kurapeev, D., Popov, M., Sidorova, M., Minasian, S., Galagudza, M., et al. (2016). Heparinase treatment of heparin-contaminated plasma from coronary artery bypass grafting patients enables reliable quantification of microRNAs. Biomolecular Detectection and Quantification, 8, 9–14. https://doi.org/10.1016/j.bdq.2016.03.001.

    Article  CAS  Google Scholar 

  12. Li, S., Zhang, F., Cui, Y., Wu, M., Lee, C., Song, J., et al. (2017). Modified high-throughput quantification of plasma microRNAs in heparinized patients with coronary artery disease using heparinase. Biochemical and Biophysical Research Communications, 493(1), 556–561. https://doi.org/10.1016/j.bbrc.2017.08.153.

    Article  CAS  PubMed  Google Scholar 

  13. Wu, C. S., Lin, F. C., Chen, S. J., Chen, Y. L., Chung, W. J., & Cheng, C. I. (2016). Optimized collection protocol for plasma microRNA measurement in patients with cardiovascular disease. BioMed Research International, 2016, 2901938. https://doi.org/10.1155/2016/2901938.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kirschner, M. B., Edelman, J. J., Kao, S. C., Vallely, M. P., van Zandwijk, N., & Reid, G. (2013). The impact of hemolysis on cell-free microRNA biomarkers. Frontiers in Genetics, 4, 94. https://doi.org/10.3389/fgene.2013.00094.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Pritchard, C. C., Kroh, E., Wood, B., Arroyo, J. D., Dougherty, K. J., Miyaji, M. M., et al. (2012). Blood cell origin of circulating microRNAs: a cautionary note for cancer biomarker studies. Cancer Prevention Research (Philadelphia, Pa.), 5(3), 492–497. https://doi.org/10.1158/1940-6207.CAPR-11-0370.

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. Shah, J. S., Soon, P. S., & Marsh, D. J. (2016). Comparison of methodologies to detect low levels of hemolysis in serum for accurate assessment of serum microRNAs. PLoS One, 11(4), e0153200. https://doi.org/10.1371/journal.pone.0153200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Pritchard, C. C., Cheng, H. H., & Tewari, M. (2012). MicroRNA profiling: approaches and considerations. Nature Reviews. Genetics, 13(5), 358–369. https://doi.org/10.1038/nrg3198.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Marabita, F., de Candia, P., Torri, A., Tegner, J., Abrignani, S., & Rossi, R. L. (2016). Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR. Briefings in Bioinformatics, 17(2), 204–212. https://doi.org/10.1093/bib/bbv056.

    Article  PubMed  Google Scholar 

  21. Schwarzenbach, H., da Silva, A. M., Calin, G., & Pantel, K. (2015). Data normalization strategies for microRNA quantification. Clinical Chemistry, 61(11), 1333–1342. https://doi.org/10.1373/clinchem.2015.239459.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wylie, D., Shelton, J., Choudhary, A., & Adai, A. T. (2011). A novel mean-centering method for normalizing microRNA expression from high-throughput RT-qPCR data. BMC Research Notes, 4, 555. https://doi.org/10.1186/1756-0500-4-555.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Vila-Navarro, E., Duran-Sanchon, S., Vila-Casadesus, M., Moreira, L., Gines, A., Cuatrecasas, M., et al. (2019). Novel circulating miRNA signatures for early detection of pancreatic neoplasia. Clinical and Translational Gastroenterology, 10(4), e00029. https://doi.org/10.14309/ctg.0000000000000029.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Xue, S., Liu, D., Zhu, W., Su, Z., Zhang, L., Zhou, C., et al. (2019). Circulating miR-17-5p, miR-126-5p and miR-145-3p are novel biomarkers for diagnosis of acute myocardial infarction. Frontiers in Physiology, 10, 123. https://doi.org/10.3389/fphys.2019.00123.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Thygesen, K., Alpert, J. S., White, H. D., & Joint ESC/ACCF/AHA/WHF Task Force for the Redefinition of Myocardial Infarction. (2007). Universal definition of myocardial infarction. Journal of the American College of Cardiology, 50(22), 2173–2195. https://doi.org/10.1016/j.jacc.2007.09.011.

    Article  PubMed  Google Scholar 

  26. Chang, P. Y., Chen, C. C., Chang, Y. S., Tsai, W. S., You, J. F., Lin, G. P., et al. (2016). MicroRNA-223 and microRNA-92a in stool and plasma samples act as complementary biomarkers to increase colorectal cancer detection. Oncotarget, 7(9), 10663–10675. https://doi.org/10.18632/oncotarget.7119.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Fan, P. C., Chen, C. C., Peng, C. C., Chang, C. H., Yang, C. H., Yang, C., et al. (2019). A circulating miRNA signature for early diagnosis of acute kidney injury following acute myocardial infarction. Journal of Translational Medicine, 17(1), 139. https://doi.org/10.1186/s12967-019-1890-7.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Marzi, M. J., Montani, F., Carletti, R. M., Dezi, F., Dama, E., Bonizzi, G., et al. (2016). Optimization and standardization of circulating microRNA detection for clinical application: the miR-test case. Clinical Chemistry, 62(5), 743–754. https://doi.org/10.1373/clinchem.2015.251942.

    Article  CAS  PubMed  Google Scholar 

  29. Mao, J., Lv, Z., & Zhuang, Y. (2014). MicroRNA-23a is involved in tumor necrosis factor-alpha induced apoptosis in mesenchymal stem cells and myocardial infarction. Experimental and Molecular Pathology, 97(1), 23–30. https://doi.org/10.1016/j.yexmp.2013.11.005.

    Article  CAS  PubMed  Google Scholar 

  30. Wang, Y., Pan, X., Fan, Y., Hu, X., Liu, X., **ang, M., et al. (2015). Dysregulated expression of microRNAs and mRNAs in myocardial infarction. American Journal of Translational Research, 7(11), 2291–2304.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Aranda, R. t., Dineen, S. M., Craig, R. L., Guerrieri, R. A., & Robertson, J. M. (2009). Comparison and evaluation of RNA quantification methods using viral, prokaryotic, and eukaryotic RNA over a 10(4) concentration range. Analytical Biochemistry, 387(1), 122–127. https://doi.org/10.1016/j.ab.2009.01.003.

    Article  CAS  PubMed  Google Scholar 

  32. Bae, I. S., Chung, K. Y., Yi, J., Kim, T. I., Choi, H. S., Cho, Y. M., et al. (2015). Identification of reference genes for relative quantification of circulating microRNAs in bovine serum. PLoS One, 10(3), e0122554. https://doi.org/10.1371/journal.pone.0122554.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Occhipinti, G., Giulietti, M., Principato, G., & Piva, F. (2016). The choice of endogenous controls in exosomal microRNA assessments from biofluids. Tumour Biology, 37(9), 11657–11665. https://doi.org/10.1007/s13277-016-5164-1.

    Article  CAS  PubMed  Google Scholar 

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Funding

This work was supported by the Chang Gung Medical Foundation (CIRPG3B0043, CLRPD1J0011, and CMRPG5G0111) and the Ministry of Science and Technology of Taiwan (106-2314-B-182A-118-MY3 and 107-2320-B-182-024). The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Author notes

  1. Chia-Chun Chen and Chen-Ching Peng are co-first authors with equal contribution, and Chia-Chun Chen and Chih-Hsiang Chang are corresponding authors with equal contribution.

Authors and Affiliations

  1. Molecular Medicine Research Center, Chang Gung University, No. 259 Wen-Hwa 1st Road, Kwei-Shan, Taoyuan, 33302, Taiwan

    Chia-Chun Chen, Chen-Ching Peng & Yu-Sun Chang

  2. Department of Colorectal Surgery, Chang Gung Memorial Hospital at Linkou, No. 5 Fusing Street, Guishan District, Taoyuan City, 333, Taiwan

    Chia-Chun Chen

  3. Department of Nephrology, Kidney Research Center, Chang Gung Memorial Hospital, Linkou Medical Center, College of Medicine, Chang Gung University, No. 5 Fusing Street, Guishan District, Taoyuan City, 333, Taiwan

    Pei-Chun Fan & Chih-Hsiang Chang

  4. Graduate Institute of Clinical Medical Sciences, Chang Gung University, No. 259 Wen-Hwa 1st Road, Kwei-Shan, Taoyuan, 33302, Taiwan

    Pei-Chun Fan & Chih-Hsiang Chang

  5. Department of Cardiology, Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, 199 Tung Hwa North Road, Taipei, 105, Taiwan

    Pao-Hsien Chu

  6. Graduate Institute of Biomedical Sciences, Chang Gung University, No. 259 Wen-Hwa 1st Road, Kwei-Shan, Taoyuan, 33302, Taiwan

    Yu-Sun Chang

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Contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Chia-Chun Chen and Chen-Ching Peng. The sample collection and clinical data processing were performed by Pei-Chun Fan and Chih-Hsiang Chang. All authors commented on previous versions of the manuscript and approved the final manuscript.

Corresponding author

Correspondence to Chia-Chun Chen.

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Conflict of Interest

The authors declare that they have no conflicts of interest.

Ethical Approval and Consent to Participate

The study protocol was approved by the Institutional Review Board of Chang Gung Medical Foundation, Taipei, Taiwan. The written informed consents were obtained from the patients themselves or legal representatives.

Animal Studies

No animal studies were carried out by the authors for this article.

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Associate Editor Yihua Bei oversaw the review of this article

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Chen, CC., Peng, CC., Fan, PC. et al. Practical Procedures for Improving Detection of Circulating miRNAs in Cardiovascular Diseases. J. of Cardiovasc. Trans. Res. 13, 977–987 (2020). https://doi.org/10.1007/s12265-020-10019-2

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