Greene Whitney A, Muñiz Alberto, Plamper Mark L, Kaini Ramesh R, Wang Heuy-Ching
Ocular Trauma, U.S. Army Institute of Surgical Research, JBSA Fort Sam Houston.
Ocular Trauma, U.S. Army Institute of Surgical Research, JBSA Fort Sam Houston;
J Vis Exp. 2014 Jun 24(88):e51589. doi: 10.3791/51589.
The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.
本报告的目的是描述用于比较人诱导多能干细胞（iPS细胞）、源自人iPS细胞的视网膜色素上皮（iPS-RPE）和胎儿视网膜色素上皮的微小RNA（miRNA）谱的实验方案。这些方案包括收集RNA以供微阵列分析，以及分析微阵列数据以鉴定在三种细胞类型中差异表达的miRNA。解释了iPS细胞和胎儿视网膜色素上皮的培养方法。还描述了用于从人iPS细胞分化出视网膜色素上皮的实验方案。我们所描述的RNA提取技术经过挑选，以便最大程度地回收非常小的RNA，用于miRNA微阵列。最后，解释了微阵列数据的细胞途径和网络分析。这些技术将有助于比较三种不同细胞类型的miRNA谱。
