A dose of 2 mg/kg lipopolysaccharide (LPS) (body weight), diluted in sterile PBS, was injected intraperitoneally into WT (annotated as WT+LPS), COX-2>COX-1, COX-1>COX-2, and Reversa mice (12-16 wk; n=6 each group). An equivalent volume of PBS alone was given to another group of WT mice (Control group, annotated as WT-LPS).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from LPS-elicited macrophages using the RNeasy Mini Kit (Cat#74104, QIAGEN, Maryland, USA). The quality of the isolated RNA was checked with Agilent RNA 6000 Nano Assay kit reagents using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Samples with a RNA Integrity Number (RIN) of 7.0 or higher were used for Nanostring analysis. The RNA samples were quantified using a standard Nanodrop ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA). The RNA concentrations were then adjusted to 20 ng/µl.
Label
none
Label protocol
n/a
Hybridization protocol
Procedures were performed according to the manufacturer’s protocol (Nanostring Technologies, Seattle, WA, USA). In brief, reactions were hybridized for about 18 h at 65°, after which the products were run through the nCounter preparation station for removal of excess probes.
Scan protocol
Raw data were generated with the nCounter digital analyzer by counting individual barcodes.
Description
Reversa
Data processing
Collected data were imported into nSolver 3.0 software (Nanostring Technologies). Annotated genotypes （WT，COX-2>COX-1, COX-1>COX-2, and Reversa, n=6, each) and treatments (Control or LPS) were used to group the data. Grouped data then were normalized to the geometric means of spiked-in positive controls (controls for assay efficiency) and spiked-in negative controls (normalized for background).
