oocyte miRNA profiling by array

This study compares miRNA expression profiles in mouse oocytes as young oocytes vs aged oocytes, and growing oocytes vs small oocytes from primordial follicles. Oocytes were derived from the ovary of young (6-8 week-old) C57BL/6 mice and aged (41-43 week-old) mice and pooled according to whether they were 20 to 50 um or 60 to 80 um in diameter. Of oocytes with the diameter of more than 60um, oocyte from young mice are called "young oocytes" and those from aged mice near the end of their reproductive life span are called "aged oocytes" to analyze the miRNA profiling associated with aging. They were also each called "small oocytes" or "large oocytes" from the size of 60um so as to investigate miRNA profiling associated with growing. Total RNA from oocytes was isolated using mirVana miRNA Isolation Kit (Applied Biosystems). MiRNA expression was profiled using Agilent's Mouse miRNA Microarray Kit (G4472A) annotated against the Sanger miRBASE 10.1 database of miRNAs. This miRNA microarray was provided by Agilent Technologies (Santa Clara, CA). Each sample was run in duplicate.本研究比较了年轻卵母细胞与衰老卵母细胞、以及生长型卵母细胞与原始卵泡来源的小型卵母细胞之间的微小RNA（microRNA，miRNA）表达谱差异。实验所用卵母细胞取自6-8周龄年轻C57BL/6小鼠与41-43周龄生殖寿命末期衰老小鼠的卵巢，并根据其直径分为20~50 μm与60~80 μm两组进行混合。对于直径大于60 μm的卵母细胞，来自年轻小鼠的被命名为“年轻卵母细胞”，来自生殖寿命末期衰老小鼠的被命名为“衰老卵母细胞”，用于分析与衰老相关的miRNA表达谱；同时以60 μm为尺寸分界，将其分别称为“小型卵母细胞”与“大型卵母细胞”，以探究与卵母细胞生长发育相关的miRNA表达谱。采用mirVana miRNA分离试剂盒（Applied Biosystems）提取卵母细胞总RNA。利用安捷伦（Agilent）小鼠miRNA基因芯片试剂盒（G4472A）进行miRNA表达谱分析，该芯片以Sanger miRBASE 10.1版miRNA数据库作为探针注释依据。该miRNA基因芯片由安捷伦科技（Agilent Technologies，美国加利福尼亚州圣克拉拉市）提供。每个样本均进行双份重复检测。