Department of Genetics, University of Georgia, Athens, GA 30602, USA.
Nucleic Acids Res. 2012 Nov 1;40(20):e156. doi: 10.1093/nar/gks680. Epub 2012 Jul 19.
RNAsnap™ is a simple and novel method that recovers all intracellular RNA quantitatively (>99%), faster (<15 min) and less expensively (∼3 cents/sample) than any of the currently available RNA isolation methods. In fact, none of the bacterial RNA isolation methods, including the commercial kits, are effective in recovering all species of intracellular RNAs (76-5700 nt) with equal efficiency, which can lead to biased results in genome-wide studies involving microarray or RNAseq analysis. The RNAsnap™ procedure yields ∼60 µg of RNA from 10(8) Escherichia coli cells that can be used directly for northern analysis without any further purification. Based on a comparative analysis of specific transcripts ranging in size from 76 to 5700 nt, the RNAsnap™ method provided the most accurate measure of the relative amounts of the various intracellular RNAs. Furthermore, the RNAsnap™ RNA was successfully used in enzymatic reactions such as RNA ligation, reverse transcription, primer extension and reverse transcriptase-polymerase chain reaction, following sodium acetate/ethanol precipitation. The RNAsnap™ method can be used to isolate RNA from a wide range of Gram-negative and Gram-positive bacteria as well as yeast.
RNAsnap™ 是一种简单新颖的方法，可定量回收（>99%）所有细胞内 RNA，速度更快（<15 分钟），成本更低（∼3 美分/样本），优于目前所有可用的 RNA 分离方法。事实上，包括商业试剂盒在内的所有细菌 RNA 分离方法都不能有效地以相同的效率回收所有种类的细胞内 RNA（76-5700nt），这可能导致涉及微阵列或 RNAseq 分析的全基因组研究产生偏倚结果。RNAsnap™ 程序可从 10(8)个大肠杆菌细胞中提取约 60µg RNA，无需进一步纯化即可直接用于 northern 分析。基于对大小为 76 至 5700nt 的特定转录物的比较分析，RNAsnap™ 方法提供了各种细胞内 RNA 相对丰度的最准确测量。此外，在经醋酸钠/乙醇沉淀后，RNAsnap™ RNA 可成功用于酶反应，如 RNA 连接、逆转录、引物延伸和逆转录酶聚合酶链反应。RNAsnap™ 方法可用于从多种革兰氏阴性和革兰氏阳性细菌以及酵母中分离 RNA。
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