RNA Extraction and PCR Analysis

Total RNA was extracted as described previously [26 (link)]. The cDNA obtained from 0.5 μg total RNA was used as a template for PCR amplification. Oligonucleotide primers were designed on the basis of GenBank entries for mouse MMP-9 and β-actin. The primers were MMP-9: 5′- GCTGACTACGATAAGGACGGCA-3′ (sense) and 5′-TAGTGGTGCAGGCAGAGTAGGA-3′ (antisense); β-actin: 5′-AGAGGGAAATCGTGCGTGAC-3′ (sense) and 5′-CAATAGTGATGACCTGGCGT-3′ (anti-sense). The amplification was performed in 30 cycles (at 55 °C, 30 s; 72 °C, 1 min; and 94 °C, 30 s). PCR fragments were determined on 2% agarose 1X TAE gel containing ethidium bromide by the molecular weight markers. The β-actin, an internal reference RNA, was amplified in parallel, and cDNA amounts were standardized to equivalent β-actin mRNA levels. The images were quantified and analyzed by an UN-SCAN-IT gel version 6.1 software (Silk Scientific, Inc., Orem, UT, USA).