Sybr green detection kit

Aortic tunica intima and media tunicas were independently cryo-pulverized in liquid nitrogen using a freezer mill (model 6750 SPEX SamplePrep). Total RNA was extracted from the pulverized tissues and vSMCs using TRIzol reagent (Life Technologies) according to the manufacturer’s protocol. Reverse transcription was performed using the MMLV enzyme (Life Technologies), and real-time PCR was conducted in a LightCycler system using a SYBR green detection kit (Roche Applied Science) with specific primers (Table 1).

Total RNA was extracted from cell cultures using TRIzol reagent (Molecular Research Center Inc., Cincinnati, OH, USA). cDNA was synthesized using a Transcriptor First Strand cDNA Synthesis Kit with oligo-dT primers (Roche Diagnostics, Indianapolis, IN, USA). Then, fluorescence real-time PCR analysis was performed using a Light Cycler 480 II instrument and SYBR Green detection kit according to the manufacturer’s protocol (Roche). The relative mRNA expression levels of the target genes were calculated using mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and human 60 S acidic ribosomal protein p0 (RPLP0) as a reference, respectively. PCR primers for measuring each of the secreted factors were as follows: for mouse CXCL1/KC, 5 -GCT GGC TTC TGA CAA CAC TAT-3 and 5 -CAA GCA GAA CTG AAC TAC CAT- 3; for mouse IL6, 5-CAA TGC TCT CCT AAC AGA TAA G-3 and 5-AGG CAT AAC GCA CTA GGT-3; for mouse GAPDH, 5-GGA GAA ACC TGC CAA GTA TGA-3, 5- GCA TCG AAG GTG GAA GAG T-3; for human CXCL1, 5-GCT TGC CTC AAT CCT GCA TC-3 and 5-GGT CAG TTG GAT TTG TCA CTG T-3; for human IL6, 5-ATC TGG ATT CAA TGA GGA GAC T-3 and 5-TGT TCC TCA CTA CTC TCA AAT CTG-3; for human RPLP0, 5-GGA AAC TCT GCA TTC TCG CT-3 and 5-GCA AGT GGG AAG GTG TAA TCC-3.