RNA Isolation Methods - Diverse Biofluids. RNA Isolation Methods - Diverse Biofluids

To evaluate performance of different extra cellular RNA isolation methods in biofluids Overall design: Serum and plasma were collected from healthy adult donors, 10 female and 10 male. Three Core exRNA isolation methods (ExoRNeasy (Qiagen), miRNeasy (Qiagen), and miRCURY Biofluids (Exiqon)) were tested in all labs, and 7 methods (Exosomal RNA Purification Kit (Norgen Biotek) w/o Amicon 3K, Exosomal RNA Purification Kit (Norgen Biotek) w/ Amicon 3K, Ultra + miRNeasy, Millipore + miRNeasy, ME Kit (New England Peptide) + miRNeasy, ExoQuick+Seramir (SBI), miRCURY Exosome Isolation + miRCURY Biofluids (Exiqon)) were tested in a subset of labs. CCCM was collected from three cell types in three different labs: human embryonic stem cells (hESCs); primary rat cardiomyocytes (NRVM); and cholangiocarcinoma cells (KMBC). exRNA isolation experiments in triplicate for the supernatants from each cell type. Bile was collected from three patients with cholangiocarcinoma using four methods: ExoRNeasy; Ultra; Millipore; and ExoQuick. exRNA isolation experiments in triplicate for the bile from each donor using five methods: ExoRNeasy; miRNeasy; Exiqon; Ultra; and Millipore. Urine was collected from healthy adult donors, 10 female and 10 male (the same donors as for plasma and serum).exRNA isolation experiments in triplicate for the urine from each donor using five methods: ExoQuick; ExoRNeasy; Homebrew; Millipore; and Ultra . Small RNA sequencing libraries were constructed using the NEB Next small RNA library kit.

为评估生物流体中不同细胞外RNA（extracellular RNA, exRNA）分离方法的性能，实验整体设计如下：从健康成年志愿者中采集血清与血浆样本，其中女性、男性各10名。3种核心细胞外RNA分离方法（ExoRNeasy（Qiagen）、miRNeasy（Qiagen）及miRCURY Biofluids（Exiqon））在所有参与实验室中开展测试；另有7种方法在部分实验室进行验证，分别为：不含Amicon 3K柱的外泌体RNA纯化试剂盒（Norgen Biotek）、含Amicon 3K柱的外泌体RNA纯化试剂盒（Norgen Biotek）、Ultra + miRNeasy、Millipore + miRNeasy、ME Kit（New England Peptide）+ miRNeasy、ExoQuick+Seramir（SBI）以及miRCURY Exosome Isolation + miRCURY Biofluids（Exiqon）。从3个不同实验室的3种细胞类型中采集细胞条件培养基（CCCM）：人胚胎干细胞（human embryonic stem cells, hESCs）、原代大鼠心肌细胞（NRVM）以及胆管癌细胞（KMBC）；针对每种细胞的培养上清液，均开展3次重复的细胞外RNA分离实验。从3名胆管癌患者体内采集胆汁样本，使用4种方法进行分离处理：ExoRNeasy、Ultra、Millipore及ExoQuick；针对每名患者的胆汁样本，采用5种方法开展3次重复的细胞外RNA分离实验，分别为ExoRNeasy、miRNeasy、Exiqon、Ultra及Millipore。尿液样本采集自与血清、血浆样本相同的健康成年志愿者，女性、男性各10名；针对每名志愿者的尿液样本，采用5种方法开展3次重复的细胞外RNA分离实验，分别为ExoQuick、ExoRNeasy、自制分离方法（Homebrew）、Millipore及Ultra。本实验使用NEB Next小RNA文库构建试剂盒完成小RNA测序文库的构建。