MicroRNA profiling of human pancreatic alpha and beta cells

MicroRNAs (miRNAs) are non-coding RNAs that play a fundamental role in regulation of gene expression affecting differentiation and development. In particular, miRNAs have been described to regulate genes important for pancreatic development and islet function. The aim of this work was to determine the miRNA expression signature in human pancreatic alpha and beta cells. miRNA stability to fixation allowed the study of microRNA in pure populations of human alpha and beta cells sorted by FACS after intracellular staining with glucagon and insulin, respectively. The determination of the specific group of miRNAs expressed in the human pancreatic alpha and beta cells may further the understanding of gene expression regulation of the islet differentiation process. The alpha and beta cells come from 6 different preparations of human pancreatic islets from donors. In this study we define expression profiles of a total of 665 miRNAs for pancreatic alpha and beta cells. For this purpose, cells were fixed with paraformaldehyde, 7AAD was applied to exclude dead cells. Then, cells were sorted after intracellular staining with C peptide to detect beta cells and glucagon to detect alpha cells. After sorting, we confirmed enriched beta cells have a purity of on average over 98%. Enriched alpha cells have a purity of on average over 98%. To determine the miRNA expression profiles, we used human miRNA TLDAs version 2. For each sample card A and card B were run after cDNA synthesis and 12 cycles of preamplification according to the manufacturer protocol. Each TLDA card A contains 1 probe for the endogenous control RNU48 while each TLDA card B contains 4 replicates of the RNU48 probe. Analysis of these controls allows calculating the intra- and inter-assay variation. Quantitative values (RQ) were calculated measuring the ddCt between the Ct values of each miRNA and the Ct value of the small nucleolar RNU48 RNA comparing the target sample and the control sample.

微小RNA（microRNAs, miRNAs）是一类非编码RNA，在调控影响细胞分化与发育的基因表达过程中发挥核心作用。尤为关键的是，已有研究表明miRNA可调控胰腺发育及胰岛功能相关的基因。本研究旨在明确人胰腺α细胞与β细胞中的miRNA表达特征谱。固定过程中miRNA的稳定性使得我们能够对经胰高血糖素、胰岛素分别进行胞内染色后，通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS）获得的纯合人胰腺α、β细胞群体中的miRNA进行研究。鉴定人胰腺α、β细胞中特异性表达的miRNA群体，有助于进一步理解胰岛分化过程中的基因表达调控机制。本次研究中的α、β细胞均来自6份不同供体来源的人胰岛制备物。本研究共构建了胰腺α、β细胞中总计665种miRNA的表达谱。为此，我们采用多聚甲醛固定细胞，使用7-氨基放线菌素D（7-AAD）排除死细胞。随后，通过C肽胞内染色标记β细胞、胰高血糖素胞内染色标记α细胞后，对细胞进行分选。分选完成后，我们验证得到富集的β细胞纯度平均可达98%以上，富集的α细胞纯度同样平均高于98%。为构建miRNA表达谱，我们使用了人类miRNA TaqMan低密度阵列v2（TaqMan Low-Density Arrays version 2, TLDAs v2）。按照试剂盒说明书，在完成cDNA合成与12个循环的预扩增后，对每个样本分别使用A卡与B卡进行检测。每张TLDA A卡包含1个内参RNU48的探针，而每张TLDA B卡则包含4个RNU48探针的重复孔。通过对这些内参对照的分析，可计算出批内与批间变异系数。定量值（相对定量值RQ）通过计算靶标样本与对照样本中各miRNA的Ct值与小核仁RNA RNU48的Ct值之间的ΔΔCt值获得。