Identification of differentially expressed lncRNAs in CpG ODN-activated macrophage

The innate immune system is the first line of defense against microbial pathogens. The activated innate immune system plays important roles in eliciting antimicrobial defenses. Despite the benefits of innate immune responses, excessive inflammation will cause host damage. Thus, tight regulation of these processes is required for the maintenance of immune homeostasis. Recently, a new class of long non-coding RNAs (lncRNAs) has emerged as important regulators in many physiological and pathological processes. Dysregulated lncRNAs have been found to be associated with excessive or uncontrolled inflammation. In this study, we employed a lncRNA microarray-based profiling assay to detect changes of lncRNAs at different stages of CpG ODN-induced macrophage activation. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed based on the function of mRNAs. Co-expression and network potential targeting relationship were constructed according to the microarray results and bioinformatics predictions. Our findings revealed the involvement of lncRNAs in the process of CpG ODN-induced macrophage activation. The RAW264.7 cells were treated with 1 μM ODN1826. At 0h, 4h, 8h, or 16h after stimulation, the cells were harvested for RNA isolation. Total RNA was extracted, and the quantity and quality were assessed by NanoDrop. And RNA integrity was detected by capillary electrophoresis using RNA 6000 Nano Lab-on-a-Chip kit and the Bioanalyzer 2100 . Then, the sample labeling, microarray hybridization and washing were performed based on the manufacturer’s standard protocols. In a brief, total RNA were transcribed to double strand cDNA, then synthesized into cRNA and labeled with Cy-3-dCTP. The labeled cRNAs were hybridized onto the microarray. After washing, the arrays were scanned by the Agilent Scanner G2505C (Agilent Technologies).