McDonald Marguerite K, Capasso Kathryn E, Ajit Seena K
Department of Pharmacology & Physiology, Drexel University College of Medicine.
J Vis Exp. 2013 Jun 14(76):e50294. doi: 10.3791/50294.
Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis. Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest. These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media. Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest. These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.
稳定的微小RNA（miRNA）存在于所有体液中，一些循环miRNA通过被包裹在称为外泌体的小囊泡中而免受降解。外泌体可与质膜融合，导致RNA和蛋白质转移至靶细胞。它们的生物学功能包括免疫反应、抗原呈递和细胞内通讯。通过血液递送可调节受体细胞中基因表达的miRNA为靶向干预开辟了新途径。除了提供一种递送药物或RNA治疗剂的策略外，外泌体内容物还可作为生物标志物，有助于诊断、确定治疗方案和预后。在这里，我们将描述定量分析血液和细胞培养基中分泌的外泌体中的miRNA和信使RNA（mRNA）的方法。纯化的外泌体将通过蛋白质印迹分析外泌体标志物以及针对感兴趣的mRNA进行PCR来进行表征。透射电子显微镜（TEM）和免疫金标记将用于验证外泌体的形态和完整性。将从这些外泌体中纯化总RNA，以确保我们能够研究同一样本中的mRNA和miRNA。通过生物分析仪验证RNA完整性后，我们将使用Taqman低密度阵列（TLDA）卡进行中通量定量实时PCR（qPCR）以鉴定外泌体miRNA，并对感兴趣的转录本进行基因表达研究。这些方案可用于量化患者、啮齿动物模型和细胞培养基在药理干预前后外泌体miRNA的变化。外泌体内容物因来源以及分泌外泌体的细胞的生理条件而异。这些差异可以提供有关细胞和系统如何应对压力或生理扰动的见解。我们的代表性数据显示，从小鼠血液、人类血液和人类细胞培养基中纯化的外泌体中存在的miRNA存在差异。在这里我们将描述定量分析血液和细胞培养基中分泌的外泌体中的miRNA和信使RNA（mRNA）的方法。纯化的外泌体将通过蛋白质印迹分析外泌体标志物以及针对感兴趣的mRNA进行PCR来进行表征。透射电子显微镜（TEM）和免疫金标记将用于验证外泌体的形态和完整性。将从这些外泌体中纯化总RNA，以确保我们能够研究同一样本中的mRNA和miRNA。通过生物分析仪验证RNA完整性后，我们将使用Taqman低密度阵列（TLDA）卡进行中通量定量实时PCR（qPCR）以鉴定外泌体miRNA，并对感兴趣的转录本进行基因表达研究。这些方案可用于量化患者、啮齿动物模型和细胞培养基在药理干预前后外泌体miRNA的变化。外泌体内容物因来源以及分泌外泌体的细胞的生理条件而异。这些差异可以提供有关细胞和系统如何应对压力或生理扰动的见解。我们的代表性数据显示，从小鼠血液、人类血液和人类细胞培养基中纯化的外泌体中存在的miRNA存在差异。
