Pinto Fernando Lopes, Thapper Anders, Sontheim Wolfgang, Lindblad Peter
Department of Photochemistry and Molecular Science, The Angström Laboratories, Uppsala University, Box 523, SE-75120, Uppsala, Sweden.
BMC Mol Biol. 2009 Aug 7;10:79. doi: 10.1186/1471-2199-10-79.
The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll a and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of Nostoc punctiforme ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction.
With this work we identify and explore strategies for improved and lower cost high quality RNA isolation from cyanobacteria. All the methods studied are suitable for RNA isolation and its use for downstream applications. We analyse different Trizol based protocols, introduce procedural changes and describe an alternative RNA extraction solution.
It was possible to improve purity of isolated RNA by modifying protocol procedures. Further improvements, both in RNA purity and experimental cost, were achieved by using a new extraction solution, PGTX.
基因表达研究的有效性和可重复性取决于提取的RNA质量以及基因组DNA污染程度。蓝细菌是革兰氏阴性原核生物，能合成叶绿素a并进行光合水氧化。这些生物拥有一系列会阻碍细胞裂解的次生代谢产物，在核酸分离方面带来了特殊挑战。因此，我们使用点状念珠藻ATCC 29133的NHM5菌株来比较和改进现有的基于苯酚的化学方法及RNA提取程序。
通过这项工作，我们确定并探索了从蓝细菌中改进和降低成本进行高质量RNA分离的策略。所有研究的方法都适用于RNA分离及其下游应用。我们分析了不同基于Trizol的方案，引入了程序变化并描述了一种替代的RNA提取溶液。
通过修改方案程序可以提高分离RNA的纯度。使用新的提取溶液PGTX在RNA纯度和实验成本方面都实现了进一步改进。
