Orientation and expression of methicillin-resistant Staphylococcus aureus small RNAs by direct multiplexed measurements using the nCounter of NanoString technology
Orientation and expression of methicillin-resistant Staphylococcus aureus small RNAs by direct multiplexed measurements using the nCounter of NanoString technology
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DOI：
10.1016/j.mimet.2010.12.025
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发表时间：
2011-02-01
影响因子：
2.2
通讯作者：
Francois, Patrice
中科院分区：
文献类型：
--
作者：
Beaume, Marie;Hernandez, David;Francois, Patrice
金黄色葡萄球菌是一种多功能的细菌机会主义者，负责广泛的感染。这种主要人类病原体的几个基因组已经公开了近10年，但细菌的毒力或致病性与基因组内容之间的全面联系仍然缺失。该项目旨在表征一组目前被标准自动注释算法忽略的小转录分子。我们评估了NanoString的nCounter分析系统确定我们最近用RNA测序（RNA-Seq）检测到的表达小RNA（sRNA）分子的方向和数量的能力。在细菌在丰富培养基中生长期间的5个时间点评估了大约70种小RNA的表达，包括位于致病岛中的sRNA。此外，两个提取策略进行了测试：RNA纯化柱或简单地从粗裂解物中的离液缓冲液的存在下制备。nCounter系统允许我们在一个实验中进行这64个测量，而无需任何酶促反应，从而避免了众所周知的技术偏差。我们评价了nCounter与定量RT-PCR（RT-qPCR）相比的重现性和可靠性。通过对两条编码链使用两种不同的设计，我们能够识别61个小RNA分子的编码链（95%）。总体而言，nCounter系统提供了与RNA-Seq数据完全一致的编码链鉴定。此外，表达结果也与RT-qPCR获得的结果相当。nCounter系统的灵敏度和最低要求为基因表达分析领域开辟了新的可能性，用于评估来自复杂介质（即宿主-病原体相互作用期间）的细菌转录谱，或者从纯化差的RNA开始，甚至直接从裂解的感染组织开始。(C)2010 Elsevier B. V.保留所有权利。
Staphylococcus aureus is a versatile bacterial opportunist responsible for a wide spectrum of infections. Several genomes of this major human pathogen have been publicly available for almost 10 years, but comprehensive links between virulence or epidemicity and genome content of the bacterium are still missing. This project aims at characterizing a set of small transcribed molecules currently ignored by standard automated annotation algorithms. We assessed the NanoString's nCounter Analysis System for its ability to determine the orientation and quantity of the expressed small RNA (sRNA) molecules that we recently detected with RNA-Sequencing (RNA-Seq). The expression of approximately seventy small RNAs, including sRNA localized in pathogenic islands, was assessed at 5 time points during growth of the bacterium in a rich medium. In addition, two extraction strategies were tested: RNA was either purified on columns or simply prepared from crude lysates in the presence of a chaotropic buffer. The nCounter System allowed us to perform these 64 measurements in a single experiment, without any enzymatic reaction, thus avoiding well-known technical biases. We evaluated the reproducibility and reliability of the nCounter compared to quantitative RT-PCR (RT-qPCR). By using two different designs for the two coding strands, we were able to identify the coding strand of 61 small RNA molecules (95%). Overall, the nCounter System provided an identification of the coding strand in perfect concordance with RNA-Seq data. In addition, expression results were also comparable to those obtained with RT-qPCR. The sensitivity and minimal requirements of the nCounter system open new possibilities in the field of gene expression analysis, for assessing bacterial transcript profiles from complex media (i.e. during host-pathogen interactions) or when starting from poorly purified RNA or even directly from lysed infected tissues. (C) 2010 Elsevier B.V. All rights reserved.