An improved acridine orange staining of DNA/RNA
An improved acridine orange staining of DNA/RNA
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DOI：
10.1016/j.acthis.2019.03.010
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发表时间：
2019-01-01
影响因子：
2.5
通讯作者：
Carvalho, Hernandes F.
中科院分区：
文献类型：
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作者：
Damas-Souza, Danilo M.;Nunes, Rony;Carvalho, Hernandes F.
需要新的工具来检查组织内单个细胞的代谢状态。我们提出了一种基于荧光的程序，由吖啶橙染色和固绿复染（AO-FG）组成，以提高前者对核酸的选择性（吖啶橙用不同的荧光颜色对DNA和RNA进行染色），而不受蛋白质的干扰。我们将该测试与选定的大鼠腹侧前列腺样本和 PC3 细胞中 RNA 和 DNA 相对量的生化定量进行了比较。前列腺上皮对于分泌物的产生代谢高度活跃。 AO-RNA 染色的差异被揭示并与上皮的代谢状态相关。特异性由 RNase A 证实。为了评估 AO-FG 染色与细胞代谢状态的关系，我们在不同浓度的葡萄糖中培养 PC3 细胞，并测量 RNA 和 DNA 量之间的比率。同时，对相似的培养物进行 AO-FG，染色模式与获得的生化结果密切相关 (r(2) = 0.886)。结果证实，AO 和 FG 的联合使用可用于同时检测 DNA 和 RNA，以及定量评估单个细胞的转录活性及其响应实验操作的变化。
New tools are desirable to examine the metabolic state of individual cells within tissues. We proposed a fluorescence-based procedure consisting of acridine orange staining and fast green counterstaining (AO-FG) to improve the selectivity of the former for nucleic acids (acridine orange stains both DNA and RNA with different fluorescence colors), with no interference from proteins. We compared this test with the biochemical quantification of the relative amounts of RNA and DNA in selected rat ventral prostate samples and PC3 cells. The epithelium of the prostate gland is highly active metabolically for the production of secretions. Differences in AO-RNA staining were revealed and correlated with the metabolic state of the epithelium. Specificity was confirmed by RNase A. To assess how AO-FG staining correlates with the metabolic state of the cell, we cultured PC3 cells in different concentrations of glucose and measured the ratios between the amounts of RNA and DNA. In parallel, similar cultures were subjected to AO-FG, and the staining pattern correlated closely (r(2) = 0.886) with the obtained biochemical results. The results confirmed that the combined use of AO and FG is useful for detecting DNA and RNA simultaneously, as well as for assessing quantitatively the transcriptional activity of individual cells and their changes in response to experimental manipulation.