Fromm Bastian, Harris Philip David, Bachmann Lutz
Natural History Museum, University of Oslo, PO Box 1172 Blindern, 0318 Oslo, Norway.
BMC Res Notes. 2011 Jun 29;4:217. doi: 10.1186/1756-0500-4-217.
Describing and evaluating miRNA inventories with Next Generation Sequencing is a goal of scientists from a wide range of fields. It requires high purity, high quality, and high yield RNA extractions that do not only contain abundant ribosomal RNAs but are also enriched in miRNAs. Here we compare 6 disparate and commercially available totalRNA extraction kits for their suitability for miRNA-preparations from Gyrodactylus salaris, an important but small (500 μm in length) monogenean pathogen of Norwegian Atlantic salmon (Salmo salar).
We evaluated 1 salt precipitation method (MasterPure™ Complete RNA Purification Kit, Epicentre), 2 Phenol based extraction methods (mirVana Kit, Ambion, and Trizol Plus Kit, Invitrogen), 1 paramagnetic bead extraction method (RNA Tissue kit, GeneMole) and 2 purification methods based on spin column chromatography using a proprietary resin as separation matrix (Phenol-free Total RNA Purification Kit, Amresco, and ZR MicroPrep Kit, Zymo Research). The quality of the extractions from 1, 10 and 100 individuals, respectively, was assessed in terms of totalRNA yield, RNA integrity, and smallRNA and miRNA yield. The 6 RNA extraction methods yielded considerably different total RNA extracts, with striking differences in low molecular weight RNA yield. The Phenol-free Total RNA Purification Kit (Amresco) showed the highest totalRNA yield, but the best miRNA/totalRNA ratio was obtained with the ZR MicroPrep Kit (Zymo Research). It was not possible to extract electrophoretically detectable miRNAs from Gyrodactylus salaris with the RNA Tissue Kit (GeneMole) or the Trizol Plus Kit (Invitrogen).
We present an optimized extraction protocol for single and small numbers of Gyrodactylus salaris from infected Atlantic salmon that delivers a totalRNA yield suitable for downstream next generation sequencing analyses of miRNA. Two of the six tested totalRNA kits/methods were not suitable for the extraction of miRNAs from Gyrodactylus salaris.
利用下一代测序技术描述和评估微小RNA（miRNA）文库是众多领域科学家的目标。这需要进行高纯度、高质量和高产量的RNA提取，提取的RNA不仅要含有丰富的核糖体RNA，还要富含miRNA。在此，我们比较了6种不同的市售总RNA提取试剂盒，以评估它们从鲑三代虫（Gyrodactylus salaris）中制备miRNA的适用性。鲑三代虫是挪威大西洋鲑（Salmo salar）的一种重要但体积较小（体长500微米）的单殖吸虫病原体。
我们评估了1种盐沉淀法（MasterPure™ 完全RNA纯化试剂盒，Epicentre公司）、2种基于苯酚的提取法（mirVana试剂盒，Ambion公司；Trizol Plus试剂盒，Invitrogen公司）、1种顺磁珠提取法（RNA Tissue试剂盒，GeneMole公司）以及2种基于使用专有树脂作为分离基质的离心柱色谱法的纯化法（无酚总RNA纯化试剂盒，Amresco公司；ZR MicroPrep试剂盒，Zymo Research公司）。分别从1、10和100个个体中提取的RNA，从总RNA产量、RNA完整性以及小RNA和miRNA产量方面评估提取质量。这6种RNA提取方法产生的总RNA提取物差异很大，在低分子量RNA产量上有显著差异。无酚总RNA纯化试剂盒（Amresco公司）的总RNA产量最高，但ZR MicroPrep试剂盒（Zymo Research公司）获得的miRNA/总RNA比例最佳。使用RNA Tissue试剂盒（GeneMole公司）或Trizol Plus试剂盒（Invitrogen公司）无法从鲑三代虫中提取出电泳可检测的miRNA。
我们提出了一种针对感染大西洋鲑体内单个和少量鲑三代虫的优化提取方案，该方案能提供适合下游miRNA下一代测序分析的总RNA产量。六种测试的总RNA试剂盒/方法中有两种不适合从鲑三代虫中提取miRNA。
