Analyze Diet
International journal of molecular sciences2019; 20(24); doi: 10.3390/ijms20246285

Expression Profile of the Chromosome 14 MicroRNA Cluster (C14MC) Ortholog in Equine Maternal Circulation throughout Pregnancy and Its Potential Implications.

Abstract: Equine chromosome 24 microRNA cluster (C24MC), the ortholog of human C14MC, is a pregnancy-related miRNA cluster. This cluster is believed to be implicated in embryonic, fetal, and placental development. The current study aimed to characterize the expression profile of this cluster in maternal circulation throughout equine gestation. The expression profile of miRNAs belonging to this cluster was analyzed in the serum of non-pregnant (diestrus), pregnant (25 d, 45 d, 4 mo, 6 mo, 10 mo), and postpartum mares. Among the miRNAs examined, 11 miRNAs were differentially expressed across the analyzed time-points. Four of these miRNAs (eca-miR-1247-3p, eca-miR-134-5p, eca-miR-382-5p, and eca-miR-433-3p) were found to be enriched in the serum of pregnant mares at Day 25 relative to non-pregnant mares. To further assess the accuracy of these miRNAs in differentiating pregnant (25 d) from non-pregnant mares, receiver operating characteristic (ROC) analysis was performed for each of these miRNAs, revealing that eca-miR-1247-3p and eca-miR-134-5p had the highest accuracy (AUCROC = 0.92 and 0.91, respectively; p < 0.05). Moreover, eca-miR-1247-3p, eca-miR-134-5p, eca-miR-409-3p, and eca-miR-379-5p were enriched in the serum of Day 45 pregnant mares. Among those miRNAs, eca-miR-1247-3p and eca-miR-409-3p retained the highest accuracy as shown by ROC analysis. GO analysis revealed that these miRNAs are mainly implicated in nervous system development as well as organ development. Using in situ hybridization, we localized eca-miR-409-3p in the developing embryo (25 d) and extra-embryonic membranes (25 and 45 d). In conclusion, the present study is the first to elucidate the circulating maternal profile of C24MC-associated miRNAs throughout pregnancy and to suggest that serum eca-miR-1247-3p, eca-miR-134-5p, and eca-miR-409-3p could be used as pregnancy-specific markers during early gestation (25 and 45 d). Overall, the high abundance of these embryo-derived miRNAs in the maternal circulation suggests an embryo-maternal communication during the equine early pregnancy.
Publication Date: 2019-12-13 PubMed ID: 31847075PubMed Central: PMC6941126DOI: 10.3390/ijms20246285Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research presents an examination of the behavior of a microRNA cluster in pregnant horses, investigating changes in its expression over the course of pregnancy, and looking into its possible role in the development of the embryo and placenta. Of the microRNAs in the group, four were noticeably higher during gestation, leading to the possibility they might serve as markers of early stages of pregnancy in horses.

Study Background and Aims

  • The study seeks to investigate a particular cluster of microRNAs (C24MC in horses, the equivalent of C14MC in humans) that are thought to be involved in the development of the embryo and placenta during pregnancy.
  • The goal is to profile their changes in expression over the course of gestation, using serum samples taken from mares at different stages of pregnancy and postpartum.
  • The microRNAs of interest are thought to hold potential as indicators of a horse’s gestation, with differential expression at different stages of the process.

Experimental Approach and Results

  • The researchers sampled serum from non-pregnant mares and those at various stages of pregnancy (25 days, 45 days, 4 months, 6 months, 10 months) and after pregnancy.
  • They found that four of the microRNAs (, , and ) were more abundant in the serum of pregnant mares on day 25 compared to non-pregnant mares.
  • ROC analysis was used to assess how well these microRNAs could distinguish between pregnant (25 days) and non-pregnant mares, revealing two of them as highly accurate indicators.
  • Further analysis showed a similar pattern for day 45 of pregnancy, with another pair of microRNAs showing greatest accuracy as markers according to ROC analysis.
  • In addition to this, GO analysis highlighted roles of these microRNAs in nervous system and organ development, and the researchers were able to locate one of the microRNAs in the embryo at day 25 and in extra-embryonic membranes at both day 25 and 45.

Conclusions and Implications

  • This work is the first to track the expression changes in this microRNA cluster over the course of gestation in horses.
  • It identifies a set of microRNAs that see a spike in expression in early pregnancy, suggesting they could be used as markers for this stage of gestation.
  • The association of this set with embryonic development hints at a communication mechanism between the embryo and the mother during these early stages.
  • The results of this study could therefore contribute to the understanding of developmental biology and assist in the field of veterinary medicine by providing new early pregnancy detection tools.

Cite This Article

APA
Dini P, El-Sheikh Ali H, Carossino M, C Loux S, Esteller-Vico A, E Scoggin K, Daels P, A Ball B. (2019). Expression Profile of the Chromosome 14 MicroRNA Cluster (C14MC) Ortholog in Equine Maternal Circulation throughout Pregnancy and Its Potential Implications. Int J Mol Sci, 20(24). https://doi.org/10.3390/ijms20246285

Publication

ISSN: 1422-0067
NlmUniqueID: 101092791
Country: Switzerland
Language: English
Volume: 20
Issue: 24

Researcher Affiliations

Dini, Pouya
  • Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium.
  • Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40503, USA.
El-Sheikh Ali, Hossam
  • Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40503, USA.
  • Theriogenology Department, Faculty of Veterinary Medicine, University of Mansoura, Mansoura 35516, Egypt.
Carossino, Mariano
  • Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40503, USA.
  • Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA.
C Loux, Shavahn
  • Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40503, USA.
Esteller-Vico, A
  • Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA.
E Scoggin, Kirsten
  • Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40503, USA.
Daels, Peter
  • Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium.
A Ball, Barry
  • Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40503, USA.

MeSH Terms

  • Animals
  • Chromosomes, Mammalian / metabolism
  • Circulating MicroRNA / blood
  • Female
  • Gene Expression Regulation / physiology
  • Horses
  • Multigene Family
  • Pregnancy / blood

Grant Funding

  • - / Universiteit Gent
  • - / University of Kentucky
  • - / Special Research Fund (BOF) at Ghent University, the Albert G. Clay Endowment and the Paul Mellon Postdoctoral fellowships at the University of Kentucky

Conflict of Interest Statement

The authors declare that they have no competing interests.

References

This article includes 61 references
  1. Bartel D.P.. MicroRNAs: Genomics, biogenesis, mechanism, and function.. Cell 2004;116:281–297.
    doi: 10.1016/S0092-8674(04)00045-5pubmed: 14744438google scholar: lookup
  2. Krol J., Sobczak K., Wilczynska U., Drath M., Jasinska A., Kaczynska D., Krzyzosiak W.J.. Structural features of microRNA (miRNA) precursors and their relevance to miRNA biogenesis and small interfering RNA/short hairpin RNA design.. J. Biol. Chem. 2004;279:42230–42239.
    doi: 10.1074/jbc.M404931200pubmed: 15292246google scholar: lookup
  3. 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;33:1290–1297.
    doi: 10.1093/nar/gki200pmc: PMC552951pubmed: 15741182google scholar: lookup
  4. Bueno M.J., Perez de Castro I., Malumbres M.. Control of cell proliferation pathways by microRNAs.. Cell Cycle 2008;7:3143–3148.
    doi: 10.4161/cc.7.20.6833pubmed: 18843198google scholar: lookup
  5. Ardekani A.M., Naeini M.M.. The Role of MicroRNAs in Human Diseases.. Avicenna J. Med. Biotechnol. 2010;2:161–179.
    pmc: PMC3558168pubmed: 23407304
  6. Ventura W., Koide K., Hori K., Yotsumoto J., Sekizawa A., Saito H., Okai T.. Placental expression of microRNA-17 and-19b is down-regulated in early pregnancy loss.. Eur. J. Obstet. Gynecol. Reprod. Biol. 2013;169:28–32.
    doi: 10.1016/j.ejogrb.2013.01.025pubmed: 23433743google scholar: lookup
  7. Zhao Z., Moley K.H., Gronowski A.M.. Diagnostic potential for miRNAs as biomarkers for pregnancy-specific diseases.. Clin. Biochem. 2013;46:953–960.
  8. Kotlabova K., Doucha J., Hromadnikova I.. Placental-specific microRNA in maternal circulation—Identification of appropriate pregnancy-associated microRNAs with diagnostic potential.. J. Reprod. Immunol. 2011;89:185–191.
    doi: 10.1016/j.jri.2011.02.006pubmed: 21513988google scholar: lookup
  9. Lai X., Vera J.. MicroRNA Clusters.. Encyclopedia of Systems Biology 2013:1310–1314.
  10. Weber M.J.. New human and mouse microRNA genes found by homology search.. FEBS J. 2005;272:59–73.
  11. Liang Y., Ridzon D., Wong L., Chen C.. Characterization of microRNA expression profiles in normal human tissues.. BMC Genom. 2007;8:166.
    doi: 10.1186/1471-2164-8-166pmc: PMC1904203pubmed: 17565689google scholar: lookup
  12. Ghai V., Wang K.. Recent progress toward the use of circulating microRNAs as clinical biomarkers.. Arch. Toxicol. 2016;90:2959–2978.
    doi: 10.1007/s00204-016-1828-2pubmed: 27585665google scholar: lookup
  13. Pritchard C.C., Cheng H.H., Tewari M.. MicroRNA profiling: Approaches and considerations.. Nat. Rev. Genet. 2012;13:358–369.
    doi: 10.1038/nrg3198pmc: PMC4517822pubmed: 22510765google scholar: lookup
  14. Gantier M.P., McCoy C.E., Rusinova I., Saulep D., Wang D., Xu D., Irving A.T., Behlke M.A., Hertzog P.J., Mackay F.. Analysis of microRNA turnover in mammalian cells following Dicer1 ablation.. Nucleic Acids Res. 2011;39:5692–5703.
    doi: 10.1093/nar/gkr148pmc: PMC3141258pubmed: 21447562google scholar: lookup
  15. Xourgia E., Papazafiropoulou A., Melidonis A.. Circulating microRNAs as biomarkers for diabetic neuropathy: A novel approach.. World J. Exp. Med. 2018;8:18–23.
    doi: 10.5493/wjem.v8.i3.18pmc: PMC6305524pubmed: 30596030google scholar: lookup
  16. Singh R., Ramasubramanian B., Kanji S., Chakraborty A.R., Haque S.J., Chakravarti A.. Circulating microRNAs in cancer: Hope or hype?. Cancer Lett. 2016;381:113–121.
    doi: 10.1016/j.canlet.2016.07.002pubmed: 27471105google scholar: lookup
  17. He Y., Lin J., Kong D., Huang M., Xu C., Kim T.K., Etheridge A., Luo Y., Ding Y., Wang K.. Current State of Circulating MicroRNAs as Cancer Biomarkers.. Clin. Chem. 2015;61:1138–1155.
    doi: 10.1373/clinchem.2015.241190pubmed: 26319452google scholar: lookup
  18. Zhou S.-S., Jin J.-P., Wang J.-Q., Zhang Z.-G., Freedman J.H., Zheng Y., Cai L.. miRNAS in cardiovascular diseases: Potential biomarkers, therapeutic targets and challenges.. Acta Pharmacol. Sin. 2018;39:1073–1084.
    doi: 10.1038/aps.2018.30pmc: PMC6289363pubmed: 29877320google scholar: lookup
  19. Kaneto C.M., Nascimento J.S., Prado M., Mendonca L.S.O.. Circulating miRNAs as biomarkers in cardiovascular diseases.. Eur. Rev. Med. Pharm. Sci. 2019;23:2234–2243.
    pubmed: 30915771
  20. Hackl M., Heilmeier U., Weilner S., Grillari J.. Circulating microRNAs as novel biomarkers for bone diseases—Complex signatures for multifactorial diseases?. Mol. Cell. Endocrinol. 2016;432:83–95.
    doi: 10.1016/j.mce.2015.10.015pubmed: 26525415google scholar: lookup
  21. Morales-Prieto D.M., Ospina-Prieto S., Chaiwangyen W., Schoenleben M., Markert U.R.. Pregnancy-associated miRNA-clusters.. J. Reprod. Immunol. 2013;97:51–61.
    doi: 10.1016/j.jri.2012.11.001pubmed: 23432872google scholar: lookup
  22. Ioannidis J., Donadeu F.X.. Circulating microRNA Profiles during the Bovine Oestrous Cycle.. PLoS ONE 2016;11:e0158160.
  23. Da Silveira J.C., Veeramachaneni D.N., Winger Q.A., Carnevale E.M., Bouma G.J.. Cell-secreted vesicles in equine ovarian follicular fluid contain miRNAs and proteins: A possible new form of cell communication within the ovarian follicle.. Biol. Reprod. 2012;86:71.
    doi: 10.1095/biolreprod.111.093252pubmed: 22116803google scholar: lookup
  24. Sohel M.M., Hoelker M., Noferesti S.S., Salilew-Wondim D., Tholen E., Looft C., Rings F., Uddin M.J., Spencer T.E., Schellander K.. Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence.. PLoS ONE 2013;8:e78505.
  25. Liang J., Wang S., Wang Z.. Role of microRNAs in embryo implantation.. Reprod. Biol. Endocrinol. 2017;15:90.
    doi: 10.1186/s12958-017-0309-7pmc: PMC5699189pubmed: 29162091google scholar: lookup
  26. Reza A.M.M.T., Choi Y.-J., Han S.G., Song H., Park C., Hong K., Kim J.-H.. Roles of microRNAs in mammalian reproduction: From the commitment of germ cells to peri-implantation embryos.. Biol. Rev. 2019;94:415–438.
    doi: 10.1111/brv.12459pmc: PMC7379200pubmed: 30151880google scholar: lookup
  27. Nothnick W.B.. The role of micro-RNAs in the female reproductive tract.. Reproduction 2012;143:559–576.
    doi: 10.1530/REP-11-0240pubmed: 22454533google scholar: lookup
  28. Hayder H., O’Brien J., Nadeem U., Peng C.. MicroRNAs: Crucial regulators of placental development.. Reproduction 2018;155:R259–R271.
    doi: 10.1530/REP-17-0603pubmed: 29615475google scholar: lookup
  29. Dini P., Daels P., Loux S.C., Esteller-Vico A., Carossino M., Scoggin K.E., Ball B.A.. Kinetics of the chromosome 14 microRNA cluster ortholog and its potential role during placental development in the pregnant mare.. BMC Genom. 2018;19:954.
    doi: 10.1186/s12864-018-5341-2pmc: PMC6302407pubmed: 30572819google scholar: lookup
  30. Prieto D.M.M., Markert U.R.. MicroRNAs in pregnancy.. J. Reprod. Immunol. 2011;88:106–111.
    doi: 10.1016/j.jri.2011.01.004pubmed: 21353310google scholar: lookup
  31. Tsochandaridis M., Nasca L., Toga C., Levy-Mozziconacci A.. Circulating microRNAs as clinical biomarkers in the predictions of pregnancy complications.. BioMed Res. Int. 2015;2015.
    doi: 10.1155/2015/294954pmc: PMC4325204pubmed: 25699269google scholar: lookup
  32. Cretoiu D., Xu J., Xiao J., Suciu N., Cretoiu S.M.. Circulating MicroRNAs as Potential Molecular Biomarkers in Pathophysiological Evolution of Pregnancy.. Dis. Markers 2016;2016:3851054.
    doi: 10.1155/2016/3851054pmc: PMC4967453pubmed: 27493447google scholar: lookup
  33. Ouyang Y., Mouillet J.F., Coyne C.B., Sadovsky Y.. Review: Placenta-specific microRNAs in exosomes—Good things come in nano-packages.. Placenta 2014;35:S69–S73.
  34. Gebremedhn S., Salilew-Wondim D., Hoelker M., Held-Hoelker E., Neuhoff C., Tholen E., Schellander K., Tesfaye D.. Exploring maternal serum microRNAs during early pregnancy in cattle.. Theriogenology 2018;121:196–203.
  35. Yang Q., Gu W.W., Gu Y., Yan N.N., Mao Y.Y., Zhen X.X., Wang J.M., Yang J., Shi H.J., Zhang X.. Association of the peripheral blood levels of circulating microRNAs with both recurrent miscarriage and the outcomes of embryo transfer in an in vitro fertilization process.. J. Transl. Med. 2018;16:186.
    doi: 10.1186/s12967-018-1556-xpmc: PMC6032771pubmed: 29973278google scholar: lookup
  36. Hosseini M.K., Gunel T., Gumusoglu E., Benian A., Aydinli K.. MicroRNA expression profiling in placenta and maternal plasma in early pregnancy loss.. Mol. Med. Rep. 2018;17:4941–4952.
    doi: 10.3892/mmr.2018.8530pmc: PMC5865953pubmed: 29393376google scholar: lookup
  37. Zhang S., Sun Q., Jiang X., Gao F.. Clinical significance of expression of hsa-mir-1247 and hsa-mir-1269a in ectopic pregnancy due to salpingitis.. Exp. Ther. Med. 2018;15:4901–4905.
    doi: 10.3892/etm.2018.5998pmc: PMC5952075pubmed: 29805512google scholar: lookup
  38. Kontomanolis E.N., Kalagasidou S., Fasoulakis Z.. MicroRNAs as Potential Serum Biomarkers for Early Detection of Ectopic Pregnancy.. Cureus 2018;10:e2344.
    doi: 10.7759/cureus.2344pmc: PMC5959728pubmed: 29796356google scholar: lookup
  39. Enquobahrie D.A., Abetew D.F., Sorensen T.K., Willoughby D., Chidambaram K., Williams M.A.. Placental microRNA expression in pregnancies complicated by preeclampsia.. Am. J. Obstet. Gynecol. 2011;204:178.e12–178.e21.
    doi: 10.1016/j.ajog.2010.09.004pmc: PMC3040986pubmed: 21093846google scholar: lookup
  40. Awamleh Z., Gloor G.B., Han V.K.M.. Placental microRNAs in pregnancies with early onset intrauterine growth restriction and preeclampsia: Potential impact on gene expression and pathophysiology.. BMC Med Genom. 2019;12:91.
    doi: 10.1186/s12920-019-0548-xpmc: PMC6598374pubmed: 31248403google scholar: lookup
  41. Loux S.C., Fernandes C.B., Dini P., Wang K., Wu X., Baxter D., Scoggin K.E., Troedsson M.H.T., Squires E.L., Ball B.A.. Small RNA (sRNA) expression in the chorioallantois, endometrium and serum of mares following experimental induction of placentitis.. Reprod. Fertil. Dev. 2019;31:1141–1156.
    doi: 10.1071/RD18400pubmed: 30947806google scholar: lookup
  42. Wommack J.C., Trzeciakowski J.P., Miranda R.C., Stowe R.P., Ruiz R.J.. Micro RNA clusters in maternal plasma are associated with preterm birth and infant outcomes.. PLoS ONE 2018;13:e0199029.
  43. Miura K., Miura S., Yamasaki K., Higashijima A., Kinoshita A., Yoshiura K.-I., Masuzaki H.. Identification of Pregnancy-Associated MicroRNAs in Maternal Plasma.. Clin. Chem. 2010;56:1767–1771.
    doi: 10.1373/clinchem.2010.147660pubmed: 20729298google scholar: lookup
  44. Schratt G.M., Tuebing F., Nigh E.A., Kane C.G., Sabatini M.E., Kiebler M., Greenberg M.E.. A brain-specific microRNA regulates dendritic spine development.. Nature 2006;439:283–289.
    doi: 10.1038/nature04367pubmed: 16421561google scholar: lookup
  45. Landgraf P., Rusu M., Sheridan R., Sewer A., Iovino N., Aravin A., Pfeffer S., Rice A., Kamphorst A.O., Landthaler M.. A mammalian microRNA expression atlas based on small RNA library sequencing.. Cell 2007;129:1401–1414.
    doi: 10.1016/j.cell.2007.04.040pmc: PMC2681231pubmed: 17604727google scholar: lookup
  46. Williams Z., Ben-Dov I.Z., Elias R., Mihailovic A., Brown M., Rosenwaks Z., Tuschl T.. Comprehensive profiling of circulating microRNA via small RNA sequencing of cDNA libraries reveals biomarker potential and limitations.. Proc. Natl. Acad. Sci. USA 2013;110:4255–4260.
    doi: 10.1073/pnas.1214046110pmc: PMC3600502pubmed: 23440203google scholar: lookup
  47. Klohonatz K.M., Cameron A.D., Hergenreder J.R., da Silveira J.C., Belk A.D., Veeramachaneni D.N., Bouma G.J., Bruemmer J.E.. Circulating miRNAs as Potential Alternative Cell Signaling Associated with Maternal Recognition of Pregnancy in the Mare.. Biol. Reprod. 2016;95:124.
    doi: 10.1095/biolreprod.116.142935pubmed: 27760749google scholar: lookup
  48. Urdinguio R.G., Fernandez A.F., Lopez-Nieva P., Rossi S., Huertas D., Kulis M., Liu C.G., Croce C.M., Calin G.A., Esteller M.. Disrupted microRNA expression caused by Mecp2 loss in a mouse model of Rett syndrome.. Epigenetics 2010;5:656–663.
    doi: 10.4161/epi.5.7.13055pmc: PMC3052849pubmed: 20716963google scholar: lookup
  49. Wu T., Lin Y., Xie Z.. MicroRNA-1247 inhibits cell proliferation by directly targeting ZNF346 in childhood neuroblastoma.. Biol. Res. 2018;51:13.
    doi: 10.1186/s40659-018-0162-ypmc: PMC5966945pubmed: 29793538google scholar: lookup
  50. Klein C.. Pregnancy Recognition and Implantation of the Conceptus in the Mare.. Adv. Anat. Embryol. Cell Biol. 2015;216:165–188.
    pubmed: 26450499
  51. Busk P.K.. A tool for design of primers for microRNA-specific quantitative RT-qPCR.. BMC Bioinform. 2014;15:29.
    doi: 10.1186/1471-2105-15-29pmc: PMC3922658pubmed: 24472427google scholar: lookup
  52. Dini P., Loux S.C., Scoggin K.E., Esteller-Vico A., Squires E.L., Troedsson M.H.T., Daels P., Ball B.A.. Identification of Reference Genes for Analysis of microRNA Expression Patterns in Equine Chorioallantoic Membrane and Serum.. Mol. Biotechnol. 2018;60:62–73.
    doi: 10.1007/s12033-017-0047-2pubmed: 29197992google scholar: lookup
  53. Bellingham S.A., Shambrook M., Hill A.F.. Quantitative Analysis of Exosomal miRNA via qPCR and Digital PCR.. Methods Mol. Biol. 2017;1545:55–70.
    pubmed: 27943207
  54. Salone V., Rederstorff M.. Stem-loop RT-PCR based quantification of small non-coding RNAs.. Methods Mol. Biol. 2015;1296:103–108.
    pubmed: 25791594
  55. Thomas P.D., Campbell M.J., Kejariwal A., Mi H., Karlak B., Daverman R., Diemer K., Muruganujan A., Narechania A.. PANTHER: A library of protein families and subfamilies indexed by function.. Genome Res. 2003;13:2129–2141.
    doi: 10.1101/gr.772403pmc: PMC403709pubmed: 12952881google scholar: lookup
  56. Dennis G., Sherman B.T., Hosack D.A., Yang J., Gao W., Lane H.C., Lempicki R.A.. DAVID: Database for annotation, visualization, and integrated discovery.. Genome Biol. 2003;4:R60.
    doi: 10.1186/gb-2003-4-9-r60pubmed: 12734009google scholar: lookup
  57. Wang Z., Yang B.. MicroRNA expression Detection Methods.. 2009.
  58. Vandesompele J., De Preter K., Pattyn F., Poppe B., Van Roy N., De Paepe A., Speleman F.. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.. Genome Biol. 2002;3:Research0034.
  59. Greiner M., Pfeiffer D., Smith R.. Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests.. Prev. Vet. Med. 2000;45:23–41.
    doi: 10.1016/S0167-5877(00)00115-Xpubmed: 10802332google scholar: lookup
  60. Hanley J.A., McNeil B.J.. The meaning and use of the area under a receiver operating characteristic (ROC) curve.. Radiology 1982;143:29–36.
  61. Swets J.A.. Measuring the accuracy of diagnostic systems.. Science 1988;240:1285–1293.
    doi: 10.1126/science.3287615pubmed: 3287615google scholar: lookup