Functional validation of miRNA-mRNA interactions in macrophages by inhibition/competition assays based in transient transfection
The binding of microRNA (miRNA) to the 3’ UTR of its target mRNAs leads to the mRNAs cleavage or the repression of protein synthesis. The study of miRNA interactions/modifications is essential to elucidate the cellularits function. The present protocol describes a transient transfection of exogenous single-stranded RNAs into bone marrow-derived macrophages (BMDM) to analyze miRNA inhibition/competition through RT-qPCR and/or In-cell western. In addition, we standardized a protocol for the study miRNA/mRNA interactions with the transient over-expression of double-stranded miRNA mimics together with a pmirGLO Dual-Luciferase miRNA Target Expression Vector containing the target miRNA inserted downstream of the luciferase reporter gene. These approaches can suggest potential targets for the development of therapies for several diseases. The entire procedure requires nearly 2 weeks.
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Posted 27 Mar, 2017
Functional validation of miRNA-mRNA interactions in macrophages by inhibition/competition assays based in transient transfection
Posted 27 Mar, 2017
The binding of microRNA (miRNA) to the 3’ UTR of its target mRNAs leads to the mRNAs cleavage or the repression of protein synthesis. The study of miRNA interactions/modifications is essential to elucidate the cellularits function. The present protocol describes a transient transfection of exogenous single-stranded RNAs into bone marrow-derived macrophages (BMDM) to analyze miRNA inhibition/competition through RT-qPCR and/or In-cell western. In addition, we standardized a protocol for the study miRNA/mRNA interactions with the transient over-expression of double-stranded miRNA mimics together with a pmirGLO Dual-Luciferase miRNA Target Expression Vector containing the target miRNA inserted downstream of the luciferase reporter gene. These approaches can suggest potential targets for the development of therapies for several diseases. The entire procedure requires nearly 2 weeks.
Figure 1
Figure 2
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Figure 4
Figure 5
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