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Molecular biotechnology2017; 60(1); 62-73; doi: 10.1007/s12033-017-0047-2

Identification of Reference Genes for Analysis of microRNA Expression Patterns in Equine Chorioallantoic Membrane and Serum.

Abstract: MicroRNAs (miRNAs) have important posttranscriptional regulatory abilities, and there is considerable interest in evaluating their expression patterns in different pathophysiological states. The most common method of quantifying miRNA expression is quantitative reverse transcription PCR; however, the identification of tissue-specific and species-specific reference miRNA is a prerequisite for miRNA expression analysis. Currently, no reference genes have been described for evaluating miRNA expression in equine serum and chorioallantoic membrane (CAM) during pregnancy. The aim of the present study was to characterize reference genes for normalization of miRNA expression in CAM and serum in the pregnant equine. To identify the most stable miRNAs in serum, expression of potential candidates was evaluated in serum samples from diestrous mares, pregnant mares and geldings. To identify the most stable miRNAs in CAM, expression of potential candidates was evaluated in CAM, collected from mares at 4, 6 and 10 months of pregnancy and immediately postpartum. From a previously generated miRNA sequencing dataset, two separate lists of potential reference miRNAs were identified (serum and CAM) using the NormFinder program, in addition to the commonly used small RNA normalizers, 5S rRNA and U6 snRNA. The putative reference miRNAs were selected using geNorm and NormFinder. In case of a nonsignificant correlation between the results of ranking and stability value between these two programs, ranking from BestKeeper was also included. NormFinder and geNorm consistently identified eca-miR-21-5p, eca-let-7a-5p and eca-miR-10a-5p as the three most stable reference genes for the normalization of serum miRNAs. Within CAM samples, the average ranking obtained from the ranking of NormFinder, geNorm and BestKeeper identified eca-miR-8908a-1-5p, eca-miR-369-5p and eca-miR-106a-5p as the three most stable miRNAs. These observations provide information about equine-specific reference genes that can be used for normalizing miRNAs expression patterns in CAM and serum during the equine pregnancy.
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Publication Date: 2017-12-05 PubMed ID: 29197992DOI: 10.1007/s12033-017-0047-2Google Scholar: Lookup
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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 identifies and characterizes reference genes necessary for normalizing microRNA expression in the chorioallantoic membrane (CAM) and serum of pregnant equines. With these reference genes identified, researchers can study the specific microRNA expression patterns during equine pregnancy.

Context and Methodology

  • MicroRNAs (miRNAs) play an important role in post-transcriptional regulation, and studying their expression patterns in various physiological states can provide scientists valuable insights into the cellular mechanisms at work.
  • The study aimed to identify stable miRNAs for use as reference genes that can allow for the normalization of miRNA expression in serum and CAM of the pregnant equine.
  • The candidate miRNA expressions in serum were evaluated using samples from diestrous mares, pregnant mares, and geldings, while the potential miRNA in CAM were evaluated at 4, 6, and 10 months of pregnancy and immediately after birth.
  • The study used a previously generated miRNA sequencing dataset, the NormFinder program, and the commonly used small RNA normalizers 5S rRNA and U6 snRNA to identify possible reference miRNAs for serum and CAM separately.

Results

  • Using the geNorm and NormFinder programs, the study identified eca-miR-21-5p, eca-let-7a-5p and eca-miR-10a-5p as the three most stable miRNAs that could serve as reference genes for normalization of serum miRNAs.
  • The average ranking generated via NormFinder, geNorm, and BestKeeper identified eca-miR-8908a-1-5p, eca-miR-369-5p, and eca-miR-106a-5p as the three most stable miRNAs within CAM samples. In cases of non-significant correlation between the results of ranking and stability, the ranking from BestKeeper was included.

Conclusion

  • The findings of this study have identified the potential reference genes for normalizing miRNA expression in the CAM and serum of pregnant equines.
  • This information is important for research in miRNA expression patterns during equine pregnancy. Understandably, these findings would greatly aid in equine healthcare research, better understanding the cellular processes during equine pregnancy, and to potentially facilitate the improvements in equine fertility and reproduction sciences.

Cite This Article

APA
Dini P, Loux SC, Scoggin KE, Esteller-Vico A, Squires EL, Troedsson MHT, Daels P, Ball BA. (2017). Identification of Reference Genes for Analysis of microRNA Expression Patterns in Equine Chorioallantoic Membrane and Serum. Mol Biotechnol, 60(1), 62-73. https://doi.org/10.1007/s12033-017-0047-2

Publication

Molecular biotechnology
ISSN: 1559-0305
NlmUniqueID: 9423533
Country: Switzerland
Language: English
Volume: 60
Issue: 1
Pages: 62-73

Researcher Affiliations

Dini, Pouya
  • Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.
  • Department of Reproduction, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Loux, Shavahn C
  • Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.
Scoggin, Kirsten E
  • Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.
Esteller-Vico, Alejandro
  • Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.
Squires, Edward L
  • Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.
Troedsson, Mats H T
  • Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.
Daels, Peter
  • Department of Reproduction, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
Ball, Barry A
  • Department of Veterinary Science, University of Kentucky, Lexington, KY, USA. b.a.ball@uky.edu.

MeSH Terms

  • Animals
  • Chorioallantoic Membrane / physiology
  • Female
  • Gene Expression Regulation, Developmental
  • Horses / genetics
  • MicroRNAs / blood
  • MicroRNAs / genetics
  • Pregnancy
  • RNA, Small Nuclear
  • Real-Time Polymerase Chain Reaction / methods
  • Real-Time Polymerase Chain Reaction / standards
  • Reference Standards

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Citations

This article has been cited 3 times.
  1. Dini P, El-Sheikh Ali H, Carossino M, C Loux S, Esteller-Vico A, E Scoggin K, Daels P, A Ball B. Expression Profile of the Chromosome 14 MicroRNA Cluster (C14MC) Ortholog in Equine Maternal Circulation throughout Pregnancy and Its Potential Implications.. Int J Mol Sci 2019 Dec 13;20(24).
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  2. Dini P, Daels P, Loux SC, Esteller-Vico A, Carossino M, Scoggin KE, Ball BA. Kinetics of the chromosome 14 microRNA cluster ortholog and its potential role during placental development in the pregnant mare.. BMC Genomics 2018 Dec 20;19(1):954.
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  3. Carossino M, Dini P, Kalbfleisch TS, Loynachan AT, Canisso IF, Shuck KM, Timoney PJ, Cook RF, Balasuriya UBR. Downregulation of MicroRNA eca-mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus.. J Virol 2018 May 1;92(9).
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