Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation
Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation
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DOI：
10.1016/j.mimet.2008.07.004
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发表时间：
2008-10-01
影响因子：
2.2
通讯作者：
Willis, David K.
中科院分区：
文献类型：
--
作者：
Jahn, Courtney E.;Charkowski, Amy O.;Willis, David K.
RNA完整性是成功定量哺乳动物组织RNA的关键，但不同组织所需的完整性水平不同。细菌RNA定量所需的完整性水平尚未确定。对三种RNA分离方法从难分离RNA的细菌Dickeya dadantii中提取高质量RNA的能力进行了评价。使用标准方案用Trizol进行细菌裂解，这种生物体的RNA产量一直很低。由于增加了热SDS孵育步骤，提高了细菌细胞裂解的产量，但根据RNA完整性数（RIN）确定的RNA质量较低。热SDS-Trizol法仍然存在DNA污染问题；RNA样品需要重复，严格的DNA酶处理，以减少DNA污染的水平足以成功的实时qRT-PCR。热sds -热苯酚RNA法获得最高的RNA质量，只需要两次DNA酶处理即可去除DNA。使用Agilent 2100 BioAnalyzer评估RNA完整性对于获得有意义的基因表达数据至关重要。real-time qRT-PCR分析基因表达时，RIN值低于7.0时变异较大，无统计学意义。我们发现，不同质量的RNA制剂在相对基因表达比率上存在巨大差异，并导致转录物水平定量的重大错误。这项工作为RNA的分离和质量评估提供了指导，这将对广泛细菌的基因表达研究有价值。Elsevier B.V.出版
RNA integrity is critical for successful RNA quantitation for mammalian tissues, but the level of integrity required differs among tissues. The level of integrity required for quantitation has not been determined for bacterial RNA. Three RNA isolation methods were evaluated for their ability to produce high quality RNA from Dickeya dadantii, a bacterium refractory to RNA isolation. Bacterial lysis with Trizol using standard protocols consistently gave low RNA yields with this organism. Higher yields due to improved bacterial cells lysis was achieved with an added hot SDS incubation step, but RNA quality was low as determined by the RNA Integrity Number (RIN). Contaminating DNA remained a problem with the hot SDS-Trizol method; RNA samples required repeated, rigorous DNase treatments to reduce DNA contamination to levels sufficient for successful real-time qRT-PCR. A hot SDS-hot phenol RNA method gave the highest RNA quality and required only two DNase treatments to remove DNA. The assessment of RNA integrity using the Agilent 2100 BioAnalyzer was critical for obtaining meaningful gene expression data. RIN values below 7.0 resulted in high variation and loss of statistical significance when gene expression was analyzed by real-time qRT-PCR. We found that RNA preparations of different quality yielded drastic differences in relative gene expression ratios and led to major errors in the quantification of transcript levels. This work provides guidelines for RNA isolation and quality assessment that will be valuable for gene expression studies in a wide range of bacteria. Published by Elsevier B.V.