Comparison of different bead-beating RNA extraction strategies: An optimized method for filamentous fungi
Comparison of different bead-beating RNA extraction strategies: An optimized method for filamentous fungi
复制标题
DOI：
10.1016/j.mimet.2012.01.011
复制
发表时间：
2012-03-01
影响因子：
2.2
通讯作者：
Medina, Angel
中科院分区：
文献类型：
--
作者：
Leite, Goncalo M.;Magan, Naresh;Medina, Angel
分子研究，特别是与次生代谢物基因簇活性相关的分子研究，需要能够从真菌生物质中提取优质 RNA。这通常受到丝状真菌细胞壁结构和内源性 RNase 活性的阻碍。因此，需要快速提取高质量RNA以用于微阵列和定量PCR测定的方法。本研究的目的是检查不同系统在高通量方法中的使用，以从丝状真菌中提取完整的 RNA。在提取方案的开发过程中测试了两种具有不同运动模式和速度能力的珠打系统。根据总 RNA 产量和总体 RNA 质量对它们进行评估。使用玻璃珠高速打珠并结合自动纯化方法，总 RNA 产量提高了三倍以上，而所需菌丝体量不到四分之一。此外，完整性和整体质量得到了保护，RNA 质量指标 (RQI) 数值始终 >7.5。该方法还降低了交叉污染风险，并将 RNA 处理量降至最低，同时仍能够进行多个样品处理，从而减少了从丝状真菌中获取 RNA 所需的时间。 (C) 2012 Elsevier B.V. 保留所有权利。
Molecular studies, especially in relation to the activity of secondary metabolite gene clusters, require the ability to extract good quality RNA from fungal biomass. This is often hindered by the cell wall structure and endogenous RNase activity in filamentous fungi. There is thus a need for rapid methods for the extraction of good quality RNA for use in microarrays and for quantitative PCR assays. The objective of this study was to examine the use of different systems for the high throughput method to extract intact RNA from filamentous fungi. Two bead beating systems with different motion patterns and speed capacities were tested in the development of the extraction protocol. They were evaluated based on the total RNA yield and overall RNA quality. The high speed bead beating with glass beads associated with an automated purification method gave more than three times higher total RNA yields with less than a quarter of the amount of mycelium required. Furthermore the integrity and overall quality was conserved, with RNA Quality Indicator (RQI) numbers consistently >7.5. This method also reduced cross contamination risks and kept RNA handling to a minimum while still being capable of multiple sample processing, reducing the time required to obtain RNA from filamentous fungi. (C) 2012 Elsevier B.V. All rights reserved.