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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
148031_e_method.png

Results

1. miRNA-microarray(AGO2 RIP-Chip)

148031_e_method.png

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)

148031_e_effect1-2.png

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

148031_e_effect2.png

Luciferase assay of Gene-X showed the repression of translation, which reflect on its binding status.


Conclusion


RIP-Chip technology is useful for
efficient identification of miRNA targets


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