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RiboCluster Profiler™
miRNA-mRNA paring analysis
Objective
Screening for target mRNAs of let-7a
Materials & Methods
| miRNA: | let-7a-1（duplex, 3' overhang） |
| Cell: | HEK293T |
| Antibodies: | Anti-EIF2C2（AGO2）（Human）mAb（Code No. RN003M） Mouse IgG2a（Code No. M076-3） |
| Kit: | RIP-Assay Kit for microRNA（Code No. RN1005） |
Results
1. miRNA-microarray（AGO2 RIP-Chip）
Both let-7a expression levels and the AGO2-binding ratio of let-7a were increased upon transfection（Total RNA: FC（Fold change）=98, AGO2-RIP-RNA: FC=6）. Detection of other let-7a family miRNAs was considered a cross-reaction with let-7a because of a common consensus seed sequence.
2. mRNA-microarray（AGO2 RIP-Chip）
The data of AGO2-RIP-RNA showed that the AGO2-binding ratio of Gene X, Y and Z were significantly changed（FC ≥ 6）. The binding status between AGO2 and mRNA was changed depending on expression level of let-7a-1.
3. Luciferase assay for confirmation of target mRNA candidates
Luciferase assay of Gene-X showed the repression of translation, which reflect on its binding status.
Conclusion
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- miRNA-mRNA paring analysis
- An emerging interest in RNA world
Introduction to post-transcriptional regulation of gene expression, function of non-coding RNAs and RNA-binding proteins
