Optimization of RNA isolation and leukocyte viability in canid RNA expression studies
Optimization of RNA isolation and leukocyte viability in canid RNA expression studies
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DOI：
10.1007/s12686-011-9465-9
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发表时间：
2012-03-01
影响因子：
1.1
通讯作者：
Wayne, Robert K.
中科院分区：
文献类型：
--
作者：
Oster, Marina;Pollinger, John P.;Wayne, Robert K.
对于RNA转录分析，RNA的来源和质量影响着真实基因表达的分辨率和表征。我们评估了从外周血中分离灰狼（Canis lupus） mRNA的收集和纯化方法，以最大限度地检测和定量低表达基因。我们专门研究了采集时全血中RNA的室温稳定性，随后的白细胞分离和RNA稳定以去除珠蛋白RNA，并使用市售试剂盒去除核糖体RNA （rRNA）。环境温度RNA稳定和珠蛋白RNA去除是成功的，mRNA提取产量和质量稳定，通过参考管家基因的定量(q)PCR验证。外周血中存在低水平的视紫红质基因mRNA，仅当白细胞mRNA被额外处理以去除核糖体RNA时，才能通过qPCR检测到。
For RNA transcript analysis, RNA source and quality influence the resolution and representation of true gene expression. We evaluate collection and purification methodologies for isolating gray wolf (Canis lupus) mRNA from peripheral whole blood to maximize detection and quantification of low expression genes. We specifically examine ambient-temperature stabilization of RNA in whole blood at time of collection, subsequent isolation and RNA stabilization of leukocytes to remove globin RNA, and removal of ribosomal RNA (rRNA) using commercially available kits. Ambient temperature RNA stabilization and globin RNA removal were successful with robust mRNA extraction yields and quality, verified by quantitative (q)PCR of reference housekeeping genes. Rhodopsin gene mRNA, present in low levels in peripheral blood, was only detected by qPCR when leukocyte mRNA was additionally treated for ribosomal RNA removal.