7900ht fast real time thermocycler

Cells were harvested from explant 3D collagen culture by digestion in a 1 mg/ml collagenase solution at 37 °C for 30 minutes on a shaker. Cells were washed once with HBSS and twice with PBS and total RNA isolation was performed using the miRCURY^™ RNA isolation kit (Exiqon, Woburn, MA) and the TURBO DNA-free kit (Ambion, Austin, TX) to eliminate residual genomic DNA. The level of mRNA of interest was assessed using a two-step quantitative reverse transcriptase-mediated real-time PCR (qPCR) method. Equal amount of total RNA was converted to cDNA by the high capacity cDNA reverse transcriptase kit (Applied Biosystems, Bedford, MA). Quantitative PCR was performed in a 7900HT Fast real-time thermocycler (Applied Biosystems) using the TaqMan Universal PCR master mix (Applied Biosystems) with probe/primer sets and the following thermocycler program: 95 °C for 20 seconds; 40 cycles of 95 °C for one second and 60 °C for 20 seconds. All probe/primer sets were purchased from Applied Biosystems (Mm00516023_m1 for mouse ICAM-1, Hs00164932_s1 for human ICAM-1, Mm00618004_m1 for EGFL7, Mm00445235_m1 for CXCL10, Mm00444662_m1 for CXCL11). The amplification data were collected by a Prism 7900 sequence detector and analyzed with Sequence Detection System software (Applied Biosystems). Data were normalized to murine GAPDH, and mRNA abundance was calculated using the ΔΔC_T method.