Quantitative comparison of DNA detection by GFP-lac repressor tagging, fluorescence in situ hybridization and immunostaining.
Quantitative comparison of DNA detection by GFP-lac repressor tagging, fluorescence in situ hybridization and immunostaining.
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通过GFP-LAC阻遏物标记，荧光原位杂交和免疫染色来对DNA检测进行定量比较。
DOI：
10.1186/1472-6750-7-92
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发表时间：
2007-12-20
影响因子：
3.5
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中科院分区：
文献类型：
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GFP 融合蛋白和免疫染色是广泛应用于研究细胞和细胞核三维组织的方法，后者通常还通过荧光原位杂交 (FISH) 进行研究。然而，这些检测方法的直接比较很少。我们对所有三种方法进行了定量比较。我们使用的细胞系包含 lac 操纵基因重复序列转基因阵列，这些重复序列可通过 GFP-lac 阻遏物融合蛋白检测到。因此，我们可以通过 GFP 荧光、针对 GFP 的抗体以及使用针对转基因阵列的探针进行 FISH 来检测单个细胞中的相同结构。 FISH 后重复抗 GFP 抗体检测。我们的结果表明，虽然从转基因阵列获得的所有四个信号总体上表现出定性和定量的相似性，但它们在细节上也有所不同。每种测试方法都揭示了特定的优点和缺点，在解释各自的实验结果时应考虑到这些优点和缺点。尽管需要变性步骤，但结构保留的细胞中的 FISH 信号与变性前产生的信号显示出惊人的相似性。
GFP-fusion proteins and immunostaining are methods broadly applied to investigate the three-dimensional organization of cells and cell nuclei, the latter often studied in addition by fluorescence in situ hybridization (FISH). Direct comparisons of these detection methods are scarce, however. We provide a quantitative comparison of all three approaches. We make use of a cell line that contains a transgene array of lac operator repeats which are detected by GFP-lac repressor fusion proteins. Thus we can detect the same structure in individual cells by GFP fluorescence, by antibodies against GFP and by FISH with a probe against the transgene array. Anti-GFP antibody detection was repeated after FISH. Our results show that while all four signals obtained from a transgene array generally showed qualitative and quantitative similarity, they also differed in details. Each of the tested methods revealed particular strengths and weaknesses, which should be considered when interpreting respective experimental results. Despite the required denaturation step, FISH signals in structurally preserved cells show a surprising similarity to signals generated before denaturation.