Mantikou Eleni, Bruning Oskar, Mastenbroek Sebastiaan, Repping Sjoerd, Breit Timo Markus, de Jong Mark
Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; MicroArray Department and Integrative Bioinformatics Unit, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, the Netherlands.
MicroArray Department and Integrative Bioinformatics Unit, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, the Netherlands; Netherlands Bioinformatics Center, Nijmegen, the Netherlands.
Fertil Steril. 2016 Feb;105(2):511-9.e4. doi: 10.1016/j.fertnstert.2015.10.025. Epub 2015 Nov 18.
To develop a reliable, reproducible, and sensitive method for investigating gene-expression profiles from individual human oocytes.
Five commercially available protocols were investigated for their efficiency to amplify messenger RNA (mRNA) from 54 single human oocytes. Protocols resulting in sufficient yields were further validated using microarray technology. For the validation, mRNA was isolated from 25 human oocytes. To eliminate biological variation, RNA from 13 human oocytes was pooled together and split into 12 identical samples for further mRNA amplification. From 12 oocytes, mRNA was individually isolated.
University medical center and university microarray laboratory.
PATIENT(S): Couples undergoing intracytoplasmic sperm injection treatment were asked to donate their immature oocytes for research, and written informed consent was obtained in all cases. Seventy-nine human oocytes were used in total.
INTERVENTION(S): None.
MAIN OUTCOME MEASURE(S): Amplification efficiency and microarray profiles.
RESULT(S): Two of the five protocols (WT-Ovation One-Direct and Arcturus RiboAMp HS Plus) resulted in sufficient yields and high success rates and were further validated for their performance in obtaining reliable, reproducible, and sensitive expression profiles from individual human oocytes. Evaluation of these two protocols demonstrated that they both displayed low technical variation and produced highly reproducible profiles (r ≥ 0.95). One of them identified significantly more transcripts but also had a higher number of false discoveries.
CONCLUSION(S): Two protocols generated ample amounts of mRNA for (quantitative) polymerase chain reaction, microarray, and sequencing techniques. Further validation using a design that discriminates between biological and technical variation showed that both protocols can be used for gene-expression profiling of individual human oocytes.
开发一种可靠、可重复且灵敏的方法，用于研究单个人类卵母细胞的基因表达谱。
研究了五种市售方案从54个单个人类卵母细胞中扩增信使核糖核酸（mRNA）的效率。对产量充足的方案使用微阵列技术进一步验证。为进行验证，从25个人类卵母细胞中分离mRNA。为消除生物学变异，将13个人类卵母细胞的RNA混合在一起，分成12个相同样本用于进一步的mRNA扩增。从12个卵母细胞中分别分离mRNA。
大学医学中心和大学微阵列实验室。
接受胞浆内单精子注射治疗的夫妇被要求捐赠其未成熟卵母细胞用于研究，所有病例均获得书面知情同意。共使用了79个人类卵母细胞。
无。
扩增效率和微阵列图谱。
五种方案中的两种（WT-Ovation One-Direct和Arcturus RiboAMp HS Plus）产量充足且成功率高，并进一步验证了它们从单个人类卵母细胞获得可靠、可重复且灵敏的表达谱的性能。对这两种方案的评估表明，它们均显示出低技术变异并产生高度可重复的图谱（r≥0.95）。其中一种方案鉴定出的转录本明显更多，但错误发现的数量也更高。
两种方案产生了足够量的mRNA用于（定量）聚合酶链反应、微阵列和测序技术。使用区分生物学变异和技术变异的设计进行的进一步验证表明，这两种方案均可用于单个人类卵母细胞的基因表达谱分析。
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