Dataset Information

Excessive MiR-365b-5p Maturation via N6-methyladenosine Regulates Chondrocyte Apoptosis and Phenotype by NCL/MiR-365b-5p/FHL1 Axis in Osteoarthritis

ABSTRACT: RNA pull-down assays were performed as the protocol of the PureBinding RNA-Protein pull-down Kit (Geneseed, Guangzhou, China). Specific biotinylated probes that hybridize to target pri-miR-365 were obtained from Ribobio. The probes (200pmol) targeting pri-miR-365 or control probes and RNA samples of chondrocytes were added to the pull-down system. After the mixture of RNA and probes was denaturated at 85°C for 5 minutes, and hybridized at room temperature for 2 hours, 100 µl streptavidin-coated magnetic beads were added to the mixture. Non-specifically bound RNAs were removed by hybridization, capture, and washing. The proteins that interacted with pri-miR-365 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, pri-miR-365-interacting bands were subjected to mass spectrometry (Beijing Genomics Institute, Shenzhen, China). The result was gained to establish a proteomic library.

ORGANISM(S): Homo Sapiens

SUBMITTER: Ming Fu

PROVIDER: PXD038257 | iProX | Mon Nov 21 00:00:00 GMT 2022

REPOSITORIES: iProX

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Project description:RNA pull-down assay. For the recombinant protein pull-down assays, 50 M-5g of recombinant His-tag TcRBP40 protein were bound to 100 uL of Ni-NTA resin (Qiagen) overnight at 4M-0C. 100 M-5g of total RNA from epimastigotes were incubated with the bound protein in 500 M-5l EMSA buffer at 4M-0C for 2 h, in the presence of Heparine and Spermidine as competitors. Bounded and supernatant samples were separated. The bound sample was washed with the same buffer three times, soft-mixing for 10 min each. After washing, RNA present in the bound and supernatantM- fractions were purified. RNA purification and amplification: RNA was extracted using the RNeasy mini kit (Qiagen). Linearly amplified RNA (aRNA) was generated with the MessageAmpM-^YII aRNA Amplification kit (Ambion), according to the manufacturerM-^Rs manual. Microarray analysis: The microarray was constructed with 70-mer oligonucleotides. Due to the hybrid and repetitive nature of the sequenced T. cruzi strain, all coding regions (CDS) identified in the genome (version 3) were retrieved and clustered by the BLASTClust program, using parameters of 40% coverage and 75% identity. For probe design, it was used ArrayOligoSelector software (v. 3.8.1), with a parameter of 50% G+C content. Was obtained 10,359 probes for the longest T. cruzi CDS of each cluster, 393 probes corresponded to the genes of an external group (Cryptosporidium hominis) and 64 spots contained only spotting solution (SSC 3x), given 10,816 spots in total. These oligonucleotides were spotted from a 50 M-5M solution onto poly-L-lysine coated slides and cross-linked with 600 mJ UV. Each probe corresponding to the T. cruzi genes was identified according to the T. cruzi Genome Consortium annotation (www.genedb.org). We compared bound and unbound mRNA, extracted from two independent pull-down assays, in a dye-swap design including four slides. Microarray images were analyzed by Spot software (Spot). The Limma package (Smyth GK, 2004) was used for background correction by the normexp method, intra-slide normalization by the printtiploess method and inter-slide normalization by the quantile method. The results for the two intra-slide probe replicates were then averaged. The pull-down results were averaged, and probes displaying more than a two-fold difference between the bound and unbound fractions were selected, at FDR 1%.

Dataset Information

Excessive MiR-365b-5p Maturation via N6-methyladenosine Regulates Chondrocyte Apoptosis and Phenotype by NCL/MiR-365b-5p/FHL1 Axis in Osteoarthritis

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