An in situ imaging method for cellular RNA tailing and structure
An imaging method and cell technology, which can be used in biochemical equipment and methods, and the determination/inspection of microorganisms. It can solve the problems of inability to obtain subcellular structure and copy number information, and achieve a simple reaction system, accurate imaging, and high reaction efficiency. Effect
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Embodiment 1
[0076] Example 1 Imaging of in situ cellular RNA tailing using the ClickerFISH method
[0077] Using the MBA-MD-231 human breast cancer cell line as the basic model, live cells were cultured with 2 μM transcription inhibitor ActD for 1 hour, and continued to culture with 100 μM 2-EA for 1 hour without changing the medium; after washing the cells with PBS for 3 times, Fix the cells with 4% (mass / volume) paraformaldehyde at room temperature for 10 minutes, wash the cells three times with PBS, and permeabilize the cells with 0.5% (volume / volume) Triton X-100 at room temperature for 5 minutes; wash with PBS After cells 3 times, add click chemistry amplification reagents including 1 μM azide-modified priming strand, 2.5 μg / mL yeast tRNA, 1 mM CuSO 4 React with 100mM sodium ascorbate at room temperature for 1 hour; wash the cells 3 times with 1xSSC, and perform a ring amplification experiment to achieve signal amplification. The specific process is as follows: First, 20μL 1xSSC hybr...
Embodiment 2
[0079] Example 2 Using the ClickerFISH method to perform RNA tailing and imaging of single-strand and double-strand structures in different cell lines
[0080]Three different cell lines of MCF-10A human normal breast epithelial cells, MCF-7 human breast cancer cells and MBA-MD-231 human breast cancer cells were selected for the following process. Live cells were cultured with 2 μM transcription inhibitor ActD for 1 hour, and continued to culture with 100 μM 2-EA for 1 hour without changing the medium; after washing the cells with PBS for 3 times, fix the cells with 4% (mass / volume) paraformaldehyde at room temperature After 10 minutes, the cells were washed 3 times with PBS, and the cells were permeabilized with 0.5% (volume / volume) Triton X-100 at room temperature for 5 minutes; after the cells were washed 3 times with PBS, the RNA single-stranded acylation reagent NAI- N3, the reaction process is: 2mMNAI-N3, 1U RNase inhibitor react with fixed cells for 30 minutes at 37°C; a...
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