Next Generation Sequencing Analysis of gene expression profile in E9.5 Mesp1Cre/+/Dgcr8-/- embryonic heart cells transfected with NC miRNA and miR-541 mimics

Purpose: To compare the E9.5 Dgcr8 conditional knockout embryonic heart cells transfected with NC miRNA and miR-541 mimics Methods: In vitro cultured E9.5 Dgcr8 conditional KO heart cells transfected with miR-541-5p and NC miRNA were extracted with TRIZOL 48hrs after transfection, and 10ng total RNA was reverse transcribed and amplified by Smart-seq2 protocol as described (Picelli et al., 2014). Duplicated biological samples were analyzed using Illumina HiSeqX10, Clean reads were mapped to mouse genome (mm9) using BWA software. Results: Genes differentially expressed in E9.5 Dgcr8 cKO embryonic heart cells transfected with NC miRNA and miR-541 were identified. Conclusions: miRNA-541 significantly changes the gene expression profiles of E9.5 Dgcr8 cKO embryonic heart cells and promote the cardiac function Overall design: Perform RNAseq of E9.5 Dgcr8 cKO embryonic heart cells transfected with NC miRNA and miR-541 mimics研究目的：比较转染阴性对照（NC）微小RNA（miRNA）与miR-541模拟物的E9.5时期Dgcr8条件性敲除（conditional knockout，cKO）胚胎心肌细胞。实验方法：将体外培养的E9.5时期Dgcr8条件性敲除心肌细胞分别转染miR-541-5p与阴性对照（NC）微小RNA，转染48小时后采用TRIZOL试剂提取细胞总RNA；取10ng总RNA，按照已发表方法（Picelli等，2014）通过Smart-seq2流程进行反转录与扩增。设置生物学重复样本，采用Illumina HiSeqX10平台进行测序；使用BWA软件将清洁读段（clean reads）比对至小鼠基因组（mm9版本）。实验结果：鉴定出转染NC miRNA与转染miR-541的E9.5时期Dgcr8条件性敲除胚胎心肌细胞之间的差异表达基因。研究结论：miR-541可显著改变E9.5时期Dgcr8条件性敲除胚胎心肌细胞的基因表达谱，并可促进心肌功能。实验整体设计：对转染NC miRNA与miR-541模拟物的E9.5时期Dgcr8条件性敲除胚胎心肌细胞开展RNA测序（RNA-seq）。