Detection and quantification of mRNA in single human polar bodies: a minimally invasive test of gene expression during oogenesis
Detection and quantification of mRNA in single human polar bodies: a minimally invasive test of gene expression during oogenesis
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DOI：
10.1093/molehr/gaq077
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发表时间：
2010-12-01
影响因子：
4
通讯作者：
Carson, Sandra A.
中科院分区：
文献类型：
--
作者：
Klatsky, Peter C.;Wessel, Gary M.;Carson, Sandra A.
卵子发生过程中产生的蛋白质和信使核糖核酸支持胚胎发育，直到受精后3天的合子过渡。由于极体可以在对卵母细胞几乎没有伤害的情况下进行活组织检查，我们检验了这样的假设，即来自减数分裂卵母细胞表达的mRNA在受精前存在并可在单个极体中检测到。人卵母细胞取自接受控制性超排卵和胞浆内单精子注射的患者。未成熟卵母细胞培养过夜，第二天检查成熟情况。对中期II卵母细胞进行极体活检，然后进行逆转录，不提取RNA。兄弟姐妹卵母细胞的制备方法类似。所有样本的互补DNA都使用选择性引物在15个循环中对候选基因进行了预扩增。实时定量聚合酶链式反应(Real-Time-PCR)检测和定量单细胞相关基因表达。在12个候选基因中有11个检测到极体mRNA。在卵母细胞中丰度较高的转录本更有可能在来自单极体的qPCR复制中被检测到。预扩增不需要提取RNA的情况下合成的c DNA，可以方便地定量检测人单个极体的m RNA。
Proteins and mRNA produced in oogenesis support embryonic development until the zygotic transition, 3 days after fertilization. Since polar bodies can be biopsied with little if any harm to the oocyte, we tested the hypothesis that mRNA originating from expression in the meiotic oocyte is present and detectable in a single polar body prior to insemination. Human oocytes were obtained from patients undergoing controlled ovarian hyperstimulation and intracytoplasmic sperm injection. Immature oocytes were cultured overnight and inspected the following day for maturation. Metaphase II oocytes underwent polar body biopsy followed by reverse transcription without RNA isolation. Sibling oocytes were similarly prepared. Complementary DNA from all samples were pre-amplified over 15 cycles for candidate genes using selective primers. Real-time PCR was performed to detect and quantify relative single-cell gene expression. Polar body mRNA was detected in 11 of 12 candidate genes. Transcripts that were present in greater abundance in the oocyte were more likely to be detected in qPCR replicates from single polar bodies. Pre-amplification of cDNA synthesized without RNA isolation can facilitate the quantitative detection of mRNA in single human polar bodies.