Rapid generation of microRNA sponges for microRNA inhibition.

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 tes...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	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
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1256f1c9cefc4fd5b38bc1b72552081e
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:1256f1c9cefc4fd5b38bc1b72552081e
record_format	dspace
spelling	oai:doaj.org-article:1256f1c9cefc4fd5b38bc1b72552081e2021-11-18T07:30:48ZRapid generation of microRNA sponges for microRNA inhibition.1932-620310.1371/journal.pone.0029275https://doaj.org/article/1256f1c9cefc4fd5b38bc1b72552081e2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22238599/?tool=EBIhttps://doaj.org/toc/1932-6203MicroRNA (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.Joost KluiverJohan H GibcusChris HettingaAnnelies AdemaMareike K S RichterNancy HalsemaIzabella Slezak-ProchazkaYe DingBart-Jan KroesenAnke van den BergPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 1, p e29275 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q 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 Rapid generation of microRNA sponges for microRNA inhibition.
description	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.
format	article
author	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
author_facet	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
author_sort	Joost Kluiver
title	Rapid generation of microRNA sponges for microRNA inhibition.
title_short	Rapid generation of microRNA sponges for microRNA inhibition.
title_full	Rapid generation of microRNA sponges for microRNA inhibition.
title_fullStr	Rapid generation of microRNA sponges for microRNA inhibition.
title_full_unstemmed	Rapid generation of microRNA sponges for microRNA inhibition.
title_sort	rapid generation of microrna sponges for microrna inhibition.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/1256f1c9cefc4fd5b38bc1b72552081e
work_keys_str_mv	AT joostkluiver rapidgenerationofmicrornaspongesformicrornainhibition AT johanhgibcus rapidgenerationofmicrornaspongesformicrornainhibition AT chrishettinga rapidgenerationofmicrornaspongesformicrornainhibition AT anneliesadema rapidgenerationofmicrornaspongesformicrornainhibition AT mareikeksrichter rapidgenerationofmicrornaspongesformicrornainhibition AT nancyhalsema rapidgenerationofmicrornaspongesformicrornainhibition AT izabellaslezakprochazka rapidgenerationofmicrornaspongesformicrornainhibition AT yeding rapidgenerationofmicrornaspongesformicrornainhibition AT bartjankroesen rapidgenerationofmicrornaspongesformicrornainhibition AT ankevandenberg rapidgenerationofmicrornaspongesformicrornainhibition
_version_	1718423331780165632

Rapid generation of microRNA sponges for microRNA inhibition.

Ejemplares similares