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兰化物复合物用于单分子光现场混合化和无背景成像

Fei Su¹, Shiyu Chen², Yuanhua Liu²

¹Institute for Biomedical Materials and Devices (IBMD), Faculty of Science, University of Technology Sydney, Sidney, NSW 2007, Australia.

Analytical chemistry

|March 6, 2024

概括

我们开发了一种基于兰化物的新探针,用于单分子光 in situ 杂交 (smFISH),可以克服敏感性问题和自光. 这种先进的smFISH探头显著提高了细胞和组织中的RNA检测.

科学领域:

生物化学生物化学
分子生物学分子生物学
生物技术是生物技术.

背景情况:

传统的单分子光在位杂交 (smFISH) 由于探针光强度低和自光,限制了RNA检测灵敏度.
挑战包括度火和组织部分的短暂自光,阻碍了清晰的信号获取.

研究的目的:

开发一种新的smFISH探头,提高灵敏度,减少RNA检测的背景噪声.
通过减轻度火和自光来解决现有的smFISH方法的局限性.

主要方法:

开发了一种新的smFISH探头,利用高度移植的兰坦化物复合物.
用于与兰坦化物复合体结合的阿齐德-dUTP的使用的奥利戈PCR.
验证了探头的稳定性,方便性和成本效益.

主要成果:

与Cy3.3相比,Lanthanide探针在SKBR3细胞中显示了2.5倍高的发光强度和3倍多的mRNA检测信号点.
在乳腺癌FFPE组织段中,HER2 mRNA检测的灵敏度比Cy3探针提高了2.7倍.
在减少背景干扰的mRNA分子成像中成功应用.

结论:

带有兰化物复合物的时间解析smFISH为核酸检测提供了一种高度敏感的方法.
开发的探头有效地克服了自流光干扰,提高了信号清晰度.
这种方法为生物样本中敏感RNA成像提供了稳定,方便和成本效益的解决方案.

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相关概念视频

FISH - Fluorescent In-situ Hybridization

FISH - Fluorescent In-situ Hybridization

Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques,...

In-situ Hybridization

In-situ Hybridization

In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled probe. The technique was first used in 1969 for the investigation of nucleic acids. It is currently an essential tool in scientific research and clinical settings, especially for diagnostic purposes.
Types of probes and labels
A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many...