Male and female mouse heart miRNA sequencing

Purpose: The goal of this study is to identify the sex differential miRNA tanscriptome between male and female adult (4-6 wk. old) mouse hearts using small RNA-seq. Methods: miRNA profiles of male and female mouse hearts were generated by sequencing, n=4 for each sex, using Illumina HiSeq2500. The sequence reads were aligned to the mm10 reference genome using STAR via the bcbio-nextgen RNA-sequencing pipeline. Differential gene expression was determined by genwiz. Results: 11 miRNAs were differentially expressed between male and female mice [ P value <0.05, |log2(Fold Change)| > 0.5], with 9 more highly expressed in female hearts and 2 more highly expressed in male hearts. Male and female adult hearts (4-6 wks), n=4 for each sex, were dissected in cold PBS and immediately homogenized in Trizol solution, and were kept in -20C until use. Small RNA from individual hearts was extracted using the miRVANA miRNA isolation kit (Thermo-Fisher AM1561). The concentration and quality of purified RNA were assessed by Tape Station (Agilent). Isolated small RNA samples were used for library prep and sequencing regardless of RIN score because small RNA bands were expected. The samples were enriched in RNAs <200nt.研究目的：本研究旨在通过小RNA测序（small RNA-seq）鉴定4-6周龄成年雌雄小鼠心脏之间的性别差异性miRNA（microRNA）转录组。实验方法：选取4-6周龄的成年雌雄小鼠心脏（每组样本量n=4），置于预冷的磷酸盐缓冲液（PBS）中解剖，随后立即在Trizol试剂中进行匀浆处理，并保存于-20℃备用。使用miRVANA miRNA分离试剂盒（Thermo-Fisher AM1561）提取单个心脏的小RNA，采用安捷伦TapeStation（Agilent）评估纯化后RNA的浓度与质量。因预期存在小RNA条带，故无论RNA完整性指数（RIN）评分如何，均使用分离得到的小RNA样本进行文库构建与测序，且所有样本均富集了长度小于200nt的RNA分子。采用Illumina HiSeq2500测序平台获取雌雄小鼠心脏的miRNA表达谱；通过bcbio-nextgen RNA测序流程结合STAR算法，将测序reads比对至mm10参考基因组；差异基因表达分析由genwiz完成。研究结果：共有11种miRNA在雌雄小鼠心脏中呈现显著性差异表达[P值<0.05，|log₂(折叠变化)|>0.5]，其中9种在雌性小鼠心脏中表达上调，2种在雄性小鼠心脏中表达上调。