基于荧光共振能量转移分子信标探针的原位杂交先进检测系统。

IF 1.6 4区 生物学 Q4 CELL BIOLOGY
Narantsog Choijookhuu, Yasuaki Shibata, Takumi Ishizuka, Yan Xu, Takehiko Koji, Yoshitaka Hishikawa
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引用次数: 4

摘要

原位杂交(ISH)是一种在细胞水平上检测特异性rna的有效方法。尽管使用半抗原标记探针的传统ISH可用于检测多种rna,但检测过程仍然复杂且需要较长的时间。因此,我们介绍了基于荧光共振能量转移(FRET)的分子信标(MB)探针在ISH中的新应用。用MCF-7细胞和C57BL/6J小鼠子宫进行ISH。ERα mRNA和28S rRNA的MB探针用Cy3/BHQ-2和6-FAM/DABCYL标记,常规探针用地高辛标记。荧光测量显示,与传统探针相比,杂交动力学更快。在MCF-7细胞中,所有细胞的核仁和细胞质中均检测到28S rRNA,而部分核仁中检测到ERα mRNA。在子宫中,利用互补MB探针可以清楚地检测到28S rRNA,而在对照载玻片中没有信号。所有细胞均检测到28S rRNA,而ERα mRNA主要在上皮细胞中检测到。28S rRNA在1个或2个碱基错配序列中荧光强度明显降低,表明对目标rna的检测具有高度特异性。综上所述,基于fret的MB探针对ISH非常有用,具有快速杂交,高灵敏度和特异性的特点。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

An Advanced Detection System for <i>In Situ</i> Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe.

An Advanced Detection System for <i>In Situ</i> Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe.

An Advanced Detection System for <i>In Situ</i> Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe.

An Advanced Detection System for In Situ Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe.

In situ hybridization (ISH) is a powerful method for detecting specific RNAs at the cellular level. Although conventional ISH using hapten-labeled probes are useful for detecting multiple RNAs, the detection procedures are still complex and required longer time. Therefore, we introduced a new application of fluorescence resonance energy transfer (FRET)-based molecular beacon (MB) probes for ISH. MCF-7 cells and C57BL/6J mouse uterus were used for ISH. MB probes for ERα mRNA and 28S rRNA were labeled with Cy3/BHQ-2 and 6-FAM/DABCYL, and conventional probes were labeled with digoxigenin. Fluorescence measurements revealed that of more-rapid hybridization kinetics compared to conventional probes. In MCF-7 cells, 28S rRNA was detected in nucleolus and cytoplasm of all cells, whereas ERα mRNA was detected in some nucleolus. In the uterus, 28S rRNA was clearly detected using complementary MB probe, but there were no signals in control slides. Moreover, 28S rRNA was detected in all cells, whereas ERα mRNA was detected mainly in the epithelium. Fluorescence intensity of 28S rRNA was decreased significantly in 1 or 2 base-mismatched sequences, that indicates highly specific detection of target RNAs. In conclusion, the FRET-based MB probes are very useful for ISH, providing rapid hybridization, high sensitivity and specificity.

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来源期刊
Acta Histochemica Et Cytochemica
Acta Histochemica Et Cytochemica 生物-细胞生物学
CiteScore
3.50
自引率
8.30%
发文量
17
审稿时长
>12 weeks
期刊介绍: Acta Histochemica et Cytochemica is the official online journal of the Japan Society of Histochemistry and Cytochemistry. It is intended primarily for rapid publication of concise, original articles in the fields of histochemistry and cytochemistry. Manuscripts oriented towards methodological subjects that contain significant technical advances in these fields are also welcome. Manuscripts in English are accepted from investigators in any country, whether or not they are members of the Japan Society of Histochemistry and Cytochemistry. Manuscripts should be original work that has not been previously published and is not being considered for publication elsewhere, with the exception of abstracts. Manuscripts with essentially the same content as a paper that has been published or accepted, or is under consideration for publication, will not be considered. All submitted papers will be peer-reviewed by at least two referees selected by an appropriate Associate Editor. Acceptance is based on scientific significance, originality, and clarity. When required, a revised manuscript should be submitted within 3 months, otherwise it will be considered to be a new submission. The Editor-in-Chief will make all final decisions regarding acceptance.
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