Analysis and Identification of Tumorigenic Targets of MicroRNA in Cancer Cells by Photoreactive Chemical Probes
Abstract
:1. Introduction
2. Results and Discussion
3. Materials and Methods
3.1. Preparation of Psoralen Modified miRNA Probes
3.2. MiR Probe-Mediated Crosslinking and RNA-Seq Analysis
3.3. Dual Luciferase Assay
3.4. Western Blotting Assay
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Protocol | PCP-Seq | CLIP [11,12] | HITS-CLIP [13] | PAR-CLIP [14,15] | CLASH [16,17] |
---|---|---|---|---|---|
UV radiation | 360 nm | 254 nm | 254 nm | 360 nm | 254 nm |
Photoactivatable nucleosides | ✓ | ✕ | ✕ | ✓ | ✕ |
Total RNA isolation | ✓ | ✕ | ✕ | ✕ | ✕ |
Partial RNase digestion | ✕ | ✓ | ✓ | ✓ | ✓ |
Creation of recombinant protein | ✕ | ✕ | ✕ | ✕ | ✓ |
Generation of stable cell line | ✕ | ✕ | ✕ | ✕ | ✓ |
Recombinant protein purification | ✕ | ✕ | ✕ | ✕ | ✓ |
Immunoprecipitation | ✕ | ✓ | ✓ | ✓ | ✕ |
Phosphatase treatment | ✕ | ✓ | ✓ | ✓ | ✓ |
3′ or 5′ RNA linker ligation | ✕ | ✓ | ✓ | ✓ | ✓ |
Radiolabeling of RNA segment | ✕ | ✓ | ✓ | ✓ | ✓ |
Purification on SDS-PAGE | ✕ | ✓ | ✓ | ✓ | ✓ |
RNA purification | ✕ | ✓ | ✓ | ✓ | ✓ |
Reverse transcription | ✓ | ✓ | ✓ | ✓ | ✓ |
PCR amplification | ✓ | ✓ | ✓ | ✓ | ✓ |
High-throughput sequencing | ✓ | ✕ | ✓ | ✓ | ✓ |
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Su, Z.; Ganbold, T.; Baigude, H. Analysis and Identification of Tumorigenic Targets of MicroRNA in Cancer Cells by Photoreactive Chemical Probes. Int. J. Mol. Sci. 2020, 21, 1545. https://doi.org/10.3390/ijms21041545
Su Z, Ganbold T, Baigude H. Analysis and Identification of Tumorigenic Targets of MicroRNA in Cancer Cells by Photoreactive Chemical Probes. International Journal of Molecular Sciences. 2020; 21(4):1545. https://doi.org/10.3390/ijms21041545
Chicago/Turabian StyleSu, Zhiyu, Tsogzolmaa Ganbold, and Huricha Baigude. 2020. "Analysis and Identification of Tumorigenic Targets of MicroRNA in Cancer Cells by Photoreactive Chemical Probes" International Journal of Molecular Sciences 21, no. 4: 1545. https://doi.org/10.3390/ijms21041545