AGO-CLIP
Argonaute-Crosslinking and Immunprecipitation
Argonaute-crosslinking and immunoprecipitation (AGO-CLIP) is a protocol that maps miRNA binding to AGO sites in C. elegans (Grosswendt et al., 2014). In this method, the photoreactive nucleoside 4-SU is first incorporated into C. elegans RNA in vivo. The 4-SU_labeled RNA is crosslinked to bound protein, and the extracted lysate is treated with RNase T1, which shortens some miRNAs. After immunoprecipitation and washing, crosslinked RNA is treated with PNK, T4 RNA ligase, and finally proteinase K. The RNA is extracted, reverse-transcribed to cDNA, and sequenced. Deep sequencing provides single base-pair resolution of miRNA and AGO binding sites.
Advantages:
- Maps miRNA binding to AGO sites using CLIP
- Provides single base-pair resolution of the binding sites
Disadvantages:
- Low efficiency in hybrid capture
- Unable to provide quantitative analysis of the strength of miRNA-target interactions (Imig et al., 2014)
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Schirle N. T., Sheu-Gruttadauria J. and MacRae I. J. Structural basis for microRNA targeting. Science. 2014;346:608-613
References:
Hamilton M. P., Rajapakshe K. I., Bader D. A., et al. The Landscape of microRNA Targeting in Prostate Cancer Defined by AGO-PAR-CLIP. Neoplasia. 2016;18:356-370
Imig J., Brunschweiger A., Brummer A., et al. miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction. Nat Chem Biol. 2015;11:107-114
Luna J. M., Scheel T. K., Danino T., et al. Hepatitis C Virus RNA Functionally Sequesters miR-122. Cell. 2015;160:1099-1110
Wang P., Ning S., Zhang Y., et al. Identification of lncRNA-associated competing triplets reveals global patterns and prognostic markers for cancer. Nucleic Acids Res. 2015;
Grosswendt S., Filipchyk A., Manzano M., et al. Unambiguous identification of miRNA:target site interactions by different types of ligation reactions. Mol Cell. 2014;54:1042-1054
Newie I., Sokilde R., Persson H., et al. The HER2-encoded miR-4728-3p regulates ESR1 through a non-canonical internal seed interaction. PLoS One. 2014;9:e97200
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History: AGO-CLIP
Revision by sbrumpton on 2017-06-21 07:50:24 - Show/Hide
Argonaute-Crosslinking and Immunprecipitation
Argonaute-crosslinking and immunoprecipitation (AGO-CLIP) is a protocol that maps miRNA binding to AGO sites in C. elegans (Grosswendt et al., 2014). In this method, the photoreactive nucleoside 4-SU is first incorporated into C. elegans RNA in vivo. The 4-SU_labeled RNA is crosslinked to bound protein, and the extracted lysate is treated with RNase T1, which shortens some miRNAs. After immunoprecipitation and washing, crosslinked RNA is treated with PNK, T4 RNA ligase, and finally proteinase K. The RNA is extracted, reverse-transcribed to cDNA, and sequenced. Deep sequencing provides single base-pair resolution of miRNA and AGO binding sites.
Advantages:- Maps miRNA binding to AGO sites using CLIP
- Provides single base-pair resolution of the binding sites
Disadvantages:- Low efficiency in hybrid capture
- Unable to provide quantitative analysis of the strength of miRNA-target interactions (Imig et al., 2014)
Reagents:Illumina Library prep and Array Kit SelectorReviews:Schirle N. T., Sheu-Gruttadauria J. and MacRae I. J. Structural basis for microRNA targeting. Science. 2014;346:608-613References:Hamilton M. P., Rajapakshe K. I., Bader D. A., et al. The Landscape of microRNA Targeting in Prostate Cancer Defined by AGO-PAR-CLIP. Neoplasia. 2016;18:356-370Imig J., Brunschweiger A., Brummer A., et al. miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction. Nat Chem Biol. 2015;11:107-114Luna J. M., Scheel T. K., Danino T., et al. Hepatitis C Virus RNA Functionally Sequesters miR-122. Cell. 2015;160:1099-1110Wang P., Ning S., Zhang Y., et al. Identification of lncRNA-associated competing triplets reveals global patterns and prognostic markers for cancer. Nucleic Acids Res. 2015;Grosswendt S., Filipchyk A., Manzano M., et al. Unambiguous identification of miRNA:target site interactions by different types of ligation reactions. Mol Cell. 2014;54:1042-1054Newie I., Sokilde R., Persson H., et al. The HER2-encoded miR-4728-3p regulates ESR1 through a non-canonical internal seed interaction. PLoS One. 2014;9:e97200