Title |
Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition
|
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Published in |
PLOS ONE, January 2012
|
DOI | 10.1371/journal.pone.0029275 |
Pubmed ID | |
Authors |
Joost Kluiver, Johan H. Gibcus, Chris Hettinga, Annelies Adema, Mareike K. S. Richter, Nancy Halsema, Izabella Slezak-Prochazka, Ye Ding, Bart-Jan Kroesen, Anke van den Berg |
Abstract |
MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies. |
X Demographics
Geographical breakdown
Country | Count | As % |
---|---|---|
Netherlands | 1 | 50% |
Australia | 1 | 50% |
Demographic breakdown
Type | Count | As % |
---|---|---|
Members of the public | 1 | 50% |
Scientists | 1 | 50% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Germany | 2 | <1% |
United States | 2 | <1% |
Netherlands | 1 | <1% |
France | 1 | <1% |
Finland | 1 | <1% |
Italy | 1 | <1% |
Egypt | 1 | <1% |
United Kingdom | 1 | <1% |
Unknown | 231 | 96% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Ph. D. Student | 69 | 29% |
Researcher | 50 | 21% |
Student > Bachelor | 25 | 10% |
Student > Master | 21 | 9% |
Student > Doctoral Student | 19 | 8% |
Other | 37 | 15% |
Unknown | 20 | 8% |
Readers by discipline | Count | As % |
---|---|---|
Agricultural and Biological Sciences | 115 | 48% |
Biochemistry, Genetics and Molecular Biology | 46 | 19% |
Medicine and Dentistry | 17 | 7% |
Neuroscience | 9 | 4% |
Engineering | 6 | 2% |
Other | 18 | 7% |
Unknown | 30 | 12% |