Figure 3. Increased expression of -defensin-1 in diseased gingival tissues. Healthy (n = 8) and inflamed (CP) (n = 8) gingival tissues were placed in RNA and, later, RNA stabilizing reagent (Quiagen, Valencia, CA, USA) and frozen at –80°C for later use. Frozen tissues were then ground, homogenized for total RNA extraction (RNeasy Kits, Quiagen), from which cDNA was synthesized (Avian RT first strand kit, Sigma, St. Louis, MO, USA). Concentration of RNA and purity of cDNA were determined by spectrophotometry. Primers used for amplification of -defensin-1 and ß-actin were designed to give an amplified product in a range of 150 to 200 bp with the use of primer3 software. Sequence of forward and backward primers is shown in the Table in Fig. 3. We performed both conventional RT-PCR and real-time PCR to determine the purity of the amplified product and quantitative expression of -defensin-1 mRNA, respectively. We quantitated -defensin-1 with ß actins as an internal control in real-time PCR. Normalized initial concentrations were then converted and represented as initial copy numbers. All quantitations were performed in triplicate. (A) Shown is the single RT-PCR amplified band of -defensin-1 (169 bp) from inflamed gingival tissue by agarose gel electrophoresis. (B) Shown is the mean copy # x 10⁶ of -defensin-1 expression with ß actins as an internal control in healthy and inflamed gingival tissues by real-time PCR. A significant (p < 0.05, Student’s t test) increase ( 10-fold) in mean copy numbers of -defensin-1 expression was observed between health and disease.