Quantitative RNA Expression Analysis

Total RNA was isolated using TRIzol reagent (Invitrogen), as per the manufacturer's instructions. TaqMan reverse transcription reagents (Applied Biosystems) were used for the synthesis of complementary DNA from total RNA, as described [22] (link), [24] (link), [25] (link). Real-time quantitative PCR was performed using SYBR Green Universal Master Mix (Applied Biosystems) and an ABI 7500 sequence detector, along with the manufacturer's software (7000v1.3.1; Applied Biosystems), as previously described [24] (link). Relative quantities of mRNAs were calculated using the comparative threshold cycle method and normalized using human cyclophilin and mouse glyceraldehydes-3-phosphate dehydrogenase (GAPDH) as an endogenous control. The primers for human IL-8 and cyclophilin, as well as, mouse MIP-2 and GAPDH were previously described [16] (link), [24] (link). The primer sequences for human and mouse MKP-1 are as follows: human MKP-1: 5′-GCTGTGCAGCAAACAGTCGA-3′ and 5′-GCCACCCTGATCGTAGAGTG-3′; mouse MKP-1: 5′-GCTGTGCAGCAAACAGTCGA-3′ and 5′-CGATTAGTCCTCATAAGGTA-3′