Total RNA from rat (n = 4, 7-days post-injury) and human (n = 5, average 10-days post-injury) blast injured tissues were prepared using the RNeasy mini Kit (Qiagen, Germantown, MD) following manufacturer’s instruction and as previously described [
24]. 500 ng of total RNA from each sample was converted into complementary DNA (cDNA) using the Superscript III First Strand Synthesis System for RT-PCR (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA). To investigate common genes related to osteogenesis and fibrosis between human and rat, real-time qRT-PCR was performed using commercially available TaqMan gene expression assays (Applied Biosystems/Thermo Fisher Scientific, Foster City, CA) for
OPN,
RUNX2 and
COL1A1 (human samples) as well as
Opn,
Runx2 and
Col1a1 (rat samples). Gene expression was normalized to
HPRT1 (human samples) and
Hprt1 (rat samples) levels, and relative gene expression was determined using the 2
−ΔΔCt method [
25]. For the cytokine gene expression profiles, RNA integrity was assessed electrophoretically using a 6000 RNA Pico lab-on-a-chip in a Bioanalyzer (Agilent, Santa Clara, CA), and cDNA was synthesized using the RT
2 First Strand Kit (SA Biosciences/Qiagen). PCR arrays were performed with a RT
2 Profiler™ PCR Array Rat Common Cytokines (cat# PARN-021Z, SA Biosciences/Qiagen) and RT
2 Profiler™ PCR Array Human Common Cytokines (cat# PAHS-021Z, SA Biosciences/Qiagen) following manufacturer’s instructions, and data analysis was performed using the RT
2 Profiler PCR Array Data Analysis software (SA Biosciences/Qiagen). All PCR-based assays were performed using an ABI7900HT system (Applied Biosystems/Thermo Fisher Scientific).