Computational protocol: miR-150 is downregulated in osteosarcoma and suppresses cell proliferation, migration and invasion by targeting ROCK1

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Protocol publication

[…] The proteins were extracted by lysing the cells in sample loading buffer, which contained 1.5% SDS, 10% glycerol, 5 mM β-mercaptoethanol, bromphenolblue and 75 mM Tris (pH 7). 15% SDS-PAGE was used to separate the whole cell lysates. Following SDS-PAGE, the proteins were transferred onto PVDF membranes. Subsequently, the membranes were incubated with appropriate primary antibodies (ROCK1, catalog no., 21850-1-AP; dilution, 1:500; ProteinTech, Inc., Wuhan, China; β-actin, catalog no., 20536-1-AP; dilution, 1:1,000) at 4°C overnight. Following incubation, specific secondary antibodies coupled to horseradish peroxidase (catalog., 15134-1-AP; dilution, 1:1,000; ProteinTech, Inc.) were used to detect the bands at room temperature. Evaluation of the expression of proteins was performed using ImageJ version 1.38 (National Institutes of Health, Bethesda, MD, USA). [...] All statistical analysis was performed using GraphPad Prism software version 5.0 (GraphPad, Software, Inc., La Jolla, CA, USA) or SigmaPlot version 12.6 (SysStat Software Inc., San Jose, CA, USA). A paired or unpaired t-test was applied to compare samples with a parametric distribution. P<0.05 was considered to indicate a statistically significant difference. Two-tailed Pearson's correlation was applied for analysis of the correlation between the expression levels of miR-150 and ROCK1. In all statistical analyses, two-sided tests were applied. All data are shown as the mean ± standard deviation. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens