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Method Article
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
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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The authors declare no competing interests exist.
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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)
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
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
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
The authors declare no competing interests exist.
Loading...
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)