NanoString nCounter gene expression panels

mRNA was isolated from each bone marrow sample using the TRIzol reagent (Invitrogen) as described in the manufacturer’s protocol. RNA samples were evaluated by nCounter gene expression analysis technology (NanoString Technologies) and quantified via nCounter Digital Analyzer (NanoString Technologies). The expression of 770 genes (including 14 internal reference genes) was determined using the nCounter Stem Cell Characterization Panel™ (mouse, NanoString, XT-CSO-MSCC-12). To minimize variability among arrays, densitometry values between arrays were normalized using Robust Multichip Average function and further transformed to a log2 scale. Gene expression levels in each sample were normalized against the geometric mean of six housekeeping genes, specifically Cltc, Gapdh, Tpia, Tbp, Pgk1, and Tubb5. A cutoff was introduced at the value of the highest negative control present on the chip. 100 ng of total mRNA was used as input and sample hybridization was performed according to the manufacturer’s instructions. Raw data processing, quality control (QC), and normalization were performed using the nSolver™ 4.0 analysis software. QC and normalization were performed with an imaging QC of >75% field of view registration, binding density QC within 0.1–2.25 range, positive control linearity QC of R2 above 0.95, and positive control limit of detection set as 0.5 fM positive control above 2 standard deviations above the mean of the negative controls. Normalization to housekeeping genes, of which genes below 100 were excluded, and pathway scoring, gene set analysis, differential expression analysis, and cell type profiling were completed using the Advanced Analysis software plugin (version 2.0.115). For each experiment, the fold changes were calculated comparing to their appropriate sham-injured saline treated control at each timepoint. For pathway scoring and differential expression analysis, a value of p of ≤0.05 was applied as cutoffs. For all NanoString analyses at 1 day, 1 week, and 1 month after injury, gene expression measurements for each group were normalized to the sham saline (n = 3) baseline of each timepoint.