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Home / Equine Research Bank / BMC Res Notes / Selection of reference genes for quantitative real-time PCR ...
BMC research notes2009; 2; 246; doi: 10.1186/1756-0500-2-246

Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts.

Abstract: Application of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between in vivo and in vitro produced embryos. Ultimately, this may lead to optimized equine assisted reproductive techniques. However, for a correct interpretation of the real-time PCR results, all data must be normalized, which is most reliably achieved by calculating the geometric mean of the most stable reference genes. In this study a set of reliable reference genes was identified for equine in vivo and fresh and frozen-thawed in vitro embryos. Results: The expression stability of 8 candidate reference genes (ACTB, GAPDH, H2A/I, HPRT1, RPL32, SDHA, TUBA4A, UBC) was determined in 3 populations of equine blastocysts (fresh in vivo, fresh and frozen-thawed in vitro embryos). Application of geNorm indicated UBC, GAPDH, ACTB and HPRT1 as the most stable genes in the in vivo embryos and UBC, RPL32, GAPDH and ACTB in both in vitro populations. When in vivo and in vitro embryos were combined, UBC, ACTB, RPL32 and GAPDH were found to be the most stable. SDHA and H2A/I appeared to be highly regulated. Conclusions: Based on these results, the geometric mean of UBC, ACTB, RPL32 and GAPDH is to be recommended for accurate normalization of quantitative real-time PCR data in equine in vivo and in vitro produced blastocysts.
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Publication Date: 2009-12-11 PubMed ID: 20003356PubMed Central: PMC2797813DOI: 10.1186/1756-0500-2-246Google 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.

This research article focuses on identifying the most reliable reference genes for normalizing quantitative real-time PCR data in equine in vivo and in vitro produced embryos. The aim is to ultimately optimize equine assisted reproductive techniques.

Objective and Methodology of the Study

  • The main objective of this study was to establish a set of reliable reference genes for equine in vivo, fresh and frozen-thawed in vitro embryos.
  • To achieve this, the researchers tested the expression stability of 8 candidate reference genes (ACTB, GAPDH, H2A/I, HPRT1, RPL32, SDHA, TUBA4A, UBC) in 3 different populations of equine blastocysts, which were fresh in vivo embryos, fresh in vitro embryos, and frozen-thawed in vitro embryos.
  • To decide on the best and most stable reference genes, the experimenters used geNorm, an algorithm that determines the most stable reference genes from a set of tested genes in a given sample panel.

Key Findings

  • The study found that UBC, GAPDH, ACTB and HPRT1 were the most stable genes in the in vivo embryos.
  • For the in vitro populations (both fresh and frozen-thawed), UBC, RPL32, GAPDH, and ACTB were the most stable genes.
  • When data from in vivo and in vitro embryos were combined, UBC, ACTB, RPL32 and GAPDH remained the most stable genes. Conversely, SDHA and H2A/I were discovered to be highly regulated.
  • The researchers concluded that the most accurate normalization of quantitative real-time PCR data in equine in vivo and in vitro produced blastocysts requires the geometric mean of UBC, ACTB, RPL32 and GAPDH.

Impact and Significance

  • By uncovering the most stable reference genes, the research has created a pathway towards enhanced accuracy in analyzing real-time PCR results in experiments involving equine in vivo and in vitro produced embryos.
  • This foundation may therefore lead to optimization of equine assisted reproductive techniques, improving fertility treatment outcomes in horses.

Cite This Article

APA
Smits K, Goossens K, Van Soom A, Govaere J, Hoogewijs M, Vanhaesebrouck E, Galli C, Colleoni S, Vandesompele J, Peelman L. (2009). Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts. BMC Res Notes, 2, 246. https://doi.org/10.1186/1756-0500-2-246

Publication

BMC research notes
ISSN: 1756-0500
NlmUniqueID: 101462768
Country: England
Language: English
Volume: 2
Pages: 246

Researcher Affiliations

Smits, Katrien
  • Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium. karen.goossens@ugent.be.
Goossens, Karen
    Van Soom, Ann
      Govaere, Jan
        Hoogewijs, Maarten
          Vanhaesebrouck, Emilie
            Galli, Cesare
              Colleoni, Silvia
                Vandesompele, Jo
                  Peelman, Luc

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