This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Functional validation of miRNA-mRNA interactions in macrophages by inhibition/competition assays based in transient transfection
Sandra Marcia Muxel
Universidade de São Paulo. Instituto de Biociências. Departamento de Fisiologia, Rua do Matão, Travessa 14, 101. São Paulo, SP, Brazil. CEP-05508-090
Corresponding Author
Lucile Maria Floeter-Winter
Universidade de São Paulo. Instituto de Biociências. Departamento de Fisiologia, Rua do Matão, Travessa 14, 101. São Paulo, SP, Brazil. CEP-05508-090
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Declarations:
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.
Keywords
Macrophage, microRNA, inhibition, competition, Real Time-qPCR, In-cell western
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Associated Publications
Leishmania (Leishmania) amazonensis induces macrophage miR-294 and miR-721 expression and modulates infection by targeting NOS2 and L-arginine metabolism
by Sandra Marcia Muxel, Maria Fernanda Laranjeira-Silva, Ricardo Andrade Zampieri, +1
Scientific Reports (08 March, 2017)
Method Article
Functional validation of miRNA-mRNA interactions in macrophages by inhibition/competition assays based in transient transfection
Sandra Marcia Muxel
Universidade de São Paulo. Instituto de Biociências. Departamento de Fisiologia, Rua do Matão, Travessa 14, 101. São Paulo, SP, Brazil. CEP-05508-090
Corresponding Author
Lucile Maria Floeter-Winter
Universidade de São Paulo. Instituto de Biociências. Departamento de Fisiologia, Rua do Matão, Travessa 14, 101. São Paulo, SP, Brazil. CEP-05508-090
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 27 Mar, 2017
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Declarations:
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
Figure 3
Figure 4
Figure 5
Associated Publications
Leishmania (Leishmania) amazonensis induces macrophage miR-294 and miR-721 expression and modulates infection by targeting NOS2 and L-arginine metabolism
by Sandra Marcia Muxel, Maria Fernanda Laranjeira-Silva, Ricardo Andrade Zampieri, +1
Scientific Reports (08 March, 2017)