Journal
JOURNAL OF DAIRY SCIENCE
Volume 104, Issue 3, Pages 3722-3735Publisher
ELSEVIER SCIENCE INC
DOI: 10.3168/jds.2020-19497
Keywords
polymerase chain reaction; methodology; embryo; blastocyst; embryo sexing
Funding
- National Institutes of Health (NIH) [R01 HD088352]
- L.E. Red Larson Endowment
- Miami Center for AIDS Research (CFAR) at the University of Miami Miller School of Medicine
- NIH [P30AI073961]
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Gene expression analysis in preimplantation embryos using a direct synthesis and specific-target pre-amplification method showed reliable results for sexing and other gene expression studies. Calibration curve analysis of PCR results validated 93.75% of genes tested, demonstrating the method's robustness. The study also showed that within-assay variation increased when cycle threshold values exceeded 18, indicating limitations in sensitivity at higher levels of gene expression.
Gene expression analysis in preimplantation embryos has been used for answering fundamental questions related to development, prediction of pregnancy outcome, and other topics. Limited amounts of mRNA in preimplantation embryos hinders progress in studying the preimplantation embryo. Here, a method was developed involving direct synthesis and specific-target pre-amplification (STA) of cDNA for gene expression analysis in single blastocysts. Effective cell lysis and genomic DNA removal steps were incorporated into the method. In addition, conditions for real-time PCR of cDNA generated from these processes were improved. By using this system, reliable embryo sexing results based on expression of sex-chromosome linked genes was demonstrated. Calibration curve analysis of PCR results using the Fluidigm Biomark microfluidic platform (Fluidigm, South San Francisco, CA) was performed to evaluate 96 STA cDNA from single blastocysts. In total, 93.75% of the genes were validated. Robust amplification was detected even when STA cDNA from a single blastocyst was diluted 1,024-fold. Further analysis showed that within-assay variation increased when cycle threshold values exceeded 18. Overall, STA quantitative real-time PCR analysis was shown to be useful for analysis of gene expression of multiple specific targets in single blastocysts.
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