Computational protocol: miRNA 132 3p inhibits osteoblast differentiation by targeting Ep300 in simulated microgravity

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Protocol publication

[…] Total mRNA was extracted using the mirVana™ miRNA Isolation Kit (Applied Biosystems, USA) according to the manufacturer’s protocol. RNA concentration was quantified using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, USA). RNA quality was verified using an Agilent 2100 bioanalyzer (Agilent Technologies, USA) and measured using the RNA integrity number (RIN). RNA samples were discarded from further analysis if their RIN scores were <5.0.miRNA microarray profiling was performed using Agilent Rat miRNA (8*15K) (Agilent Technologies) as previously described. The microarray contains probes for 677 mature rat miRNAs found in the Sanger miRBase database version V16.0. Total RNA of bone tissues from CON (n = 3) and HU (n = 3) were subjected to microarray analysis. The microarray image information was converted into spot intensity values using Scanner Control Software Rev. 7.0 (Agilent Technologies). The signal after background subtraction was exported directly into the GeneSpring GX11.0 software (Agilent Technologies) for quantile normalization and further analysis. If the expression of an mRNA changed more than 2-fold in the HU group compared with the CON group, it was submitted to hierarchical clustering analysis. [...] Computational miRNA target prediction analyses were performed using the databases TargetScan (http://www.targetscan.org/mmu_61/) and PicTar (http://pictar.mdc-berlin.de/.). The predicted genes were sorted based on the context scores, and those encoding osteogenic ossification-related extracellular signaling molecules were extracted. Sequence conservation was examined using University of California Santa Cruz genome browser (http://genome.ucsu.edu/). […]

Pipeline specifications

Software tools GeneSpring GX, TargetScan, PicTar
Databases miRBase
Applications miRNA array analysis, Genome data visualization
Organisms Rattus norvegicus
Diseases Alveolar Bone Loss