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首页> 外文期刊>Molecular biotechnology >Quantitative aspects of an in situ hybridization procedure for detecting mRNAs in cells using 96-well microplates.
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Quantitative aspects of an in situ hybridization procedure for detecting mRNAs in cells using 96-well microplates.

机译：使用96孔微孔板检测细胞中mRNA的原位杂交程序的定量方面。

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摘要

The universal quantitation of the DNA hybridization reaction has been a goal sought by many researchers. Part of this search has been the need to develop a rapid, sensitive, easy-to-perform, and quantitative method to measure the abundance of specific mRNAs directly within cells. Conventionally mRNA detection can be done by advanced quantitative in situ hybridization (ISH) using either image analysis or fluorescence in situ hybridization (FISH), or indirectly by extraction of mRNA from cells or tissue and using Northern blot or quantitative polymerase chain reaction (PCR). We examined the quantitative nature of probe binding to intracellular mRNA in a sensitive and easy-to-use nonisotopic method of ISH previously developed in our laboratories. The method is applicable to isolated primary cells or cells in culture. The procedural details are very simple, with cells being centrifuged into 96-well microplates, fixed with formalin, and pretreated with Triton X-100 and Nonidet P-40 before photobiotin-labeled cDNA probes are applied. Biotin from the hybridization of probe to target is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and visualized by the p-nitrophenyl phosphate conversion method. The quantitative parameters of the ISH procedure were determined by measuring the levels of expression of erythropoietin (EPO) mRNA and its translated protein in transfected COS-7 cells. There is a log-linear relationship between the levels of signal obtained in the ISH reaction in 96-well microplates and the EPO protein levels measured by enzyme-linked immunosorbent assay (ELISA). This demonstrated relationship is important in the standardization and use of these procedures to measure quantitatively mRNAs within cells.
机译：DNA杂交反应的通用定量已成为许多研究人员追求的目标。这种搜索的一部分是需要开发一种快速，敏感，易于执行的定量方法来直接在细胞内测量特定mRNA的丰度。常规地，可以使用图像分析或荧光原位杂交（FISH）通过高级定量原位杂交（ISH）进行mRNA检测，或通过从细胞或组织中提取mRNA并使用Northern blot或定量聚合酶链反应（PCR）间接进行mRNA检测。 。我们使用以前在我们实验室中开发的ISH敏感且易于使用的非同位素方法，研究了探针与细胞内mRNA结合的定量性质。该方法适用于分离的原代细胞或培养的细胞。程序的细节非常简单，将细胞离心成96孔微孔板，用福尔马林固定，并在应用光生物素标记的cDNA探针之前用Triton X-100和Nonidet P-40进行预处理。使用链霉亲和素和生物素化的碱性磷酸酶的多次应用，可以检测到探针与靶标杂交产生的生物素，并通过对硝基苯基磷酸酯转化法将其可视化。 ISH方法的定量参数是通过测量转染的COS-7细胞中促红细胞生成素（EPO）mRNA及其翻译蛋白的表达水平来确定的。在96孔微孔板的ISH反应中获得的信号水平与通过酶联免疫吸附测定（ELISA）测量的EPO蛋白水平之间存在对数线性关系。这种证明的关系在标准化和使用这些程序定量测量细胞内mRNA方面很重要。

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