Department of Animal Sciences, D.H. Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville 32611-0910.
Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL 33136.
J Dairy Sci. 2021 Mar;104(3):3722-3735. doi: 10.3168/jds.2020-19497. Epub 2021 Jan 15.
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 preamplification (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.
在着床前胚胎中进行基因表达分析，可用于回答与发育相关的基本问题、预测妊娠结局和其他课题。 由于着床前胚胎中 mRNA 的含量有限，因此在研究着床前胚胎方面进展缓慢。 在这里，开发了一种方法，涉及 cDNA 的直接合成和特定靶标预扩增（STA），用于单个囊胚的基因表达分析。 该方法纳入了有效的细胞裂解和基因组 DNA 去除步骤。 此外，还改进了从这些过程中生成的 cDNA 的实时 PCR 条件。 通过使用该系统，基于性染色体连锁基因的表达，成功地证明了可靠的胚胎性别鉴定结果。 使用 Fluidigm Biomark 微流控平台（Fluidigm，加利福尼亚州南旧金山）对 PCR 结果进行校准曲线分析，以评估来自单个囊胚的 96 个 STA cDNA。 总共验证了 93.75%的基因。 即使将单个囊胚的 STA cDNA 稀释 1024 倍，也能检测到稳健的扩增。 进一步的分析表明，当循环阈值超过 18 时，测定内变异性增加。 总体而言，STA 实时定量 PCR 分析可用于分析单个囊胚中多个特定靶标的基因表达。
