Project description:Here we show that biotin-labelled miR-34a can be loaded to AGO2, and AGO2 immunoprecipitation can pulldown biotinylated miR-34a (Bio-miR pulldown). RNA-sequencing (RNA-seq) of the Bio-miR pulldown RNAs efficiently identified miR-34a mRNA targets, which could be verified with luciferase assays. In contrast to the approach of Bio-miR pulldown, RNA-seq of miR-34a overexpression samples had limited value in identifying direct targets of miR-34a. It seems that pulldown of 30 -Biotin-tagged miRNA can identify bona fide microRNA targets at least for miR34a.
Project description:T-47d breast cancer cells were transfected in 6 test/control replicates with biotinylated miR-190b mimic or biotinylated mimic negative control by Qiagen. After 48h cells were lysed and miRNA:mRNA complexes were pulled-down by double precipitation. Firstly by pulling down Ago2 with Dynabeads G with rad Ago2 antibody and secondly via streptavidin coated magnetic beads. The precipitation was carried out according to manufacturer protocol. RNA from the eluted samples was then isolated using phenol-chloroform-isoamyl alcohol mixure and suspended in RNAse free water. This experiment was implemented to assess direct targets bound by miR-190b in the ER-positive breast cancer subtype T-47d.
Project description:Confluent U2OS cells were released to serum-free medium, and continued to grow for 36 h (Per2-dLuc peak), then added biotin-DRB (40uM) treatment for 0.5, then fixed the cells, harvested genomic DNA by Strepavidin agarose beads, and conducted hi-seq sequencing
Project description:We performed pulldown assays followed by mass-spectrometry analysis using biotin-tagged 13-cis retinoic acid to identify its binding targets in HeLa cells
Project description:Vectors carrying lnc-GD2H and antisense lnc-GD2H were linearized with the corresponding restriction enzymes to prepare template DNAs for in vitro transcription. Biotinylated RNAs were mixed with proteins extracted from C2C12 cells, followed by targeting of the RNAs with streptavidin beads. After SDS-PAGE following RNA pulldown, the differentially expressed proteins were enzymolyzed and used for MS analysis.
Project description:Goal was to identify RNA-binding proteins with a preference for m6A-modified RNA using an RNA bait generated from a known m6A site in the Dlg4 (PSD-95) mRNA 3`-UTR. By comparison of differential enrichment between m6A-RNA pulldown fractions and A-RNA or no RNA fractions, we can identify m6A-binding proteins.