Uvp geldoc it imaging system

cDNA was synthesized from 2 μg of total RNA using oligo (dT)_12-18 primer and Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega, Madison, WI). After cDNA synthesis, PCR amplification was carried out using appropriate sense and antisense primers specific for rat β-actin (a house-keeping gene), IL-1β, IL-6, Mob-1, KC, CD14, TLR-4, Myd88, NF-κB, I-κBα, and I-κBζ synthesized by Eurofins Genomics (Huntville, AL) in a final volume of 20 μl containing 1 μl of cDNA, 1X PCR buffer, 0.2 μM of each sense and anti-sense primer, 0.2 mM of dNTPs, and 0.5 unit of Taq DNA polymerase (Applied Biosystems, Foster City, CA) [10 (link)] [13 (link)]. The reaction was heated to 94 °C for 5 min, followed by appropriate cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, and extension at 72 °C for 30 s. After the final cycle, a 7-min extension step at 72° C was included. PCR products were then run on a 2.0% agarose gel and the gel image was recorded using a UVP GelDoc-It^™ imaging system (UVP, Upland, CA). The band intensities of genes of interest were digitized using VisionWorks^™ LS software (UVP, Upland, CA) and normalized against the intensity of β-actin in the same sample.