Figure 5
Quantification of AQP5 mRNA and AQP5 protein in ascites-derived chicken ovarian cancer (COVCAR) cells. A. Quantification of AQP5 mRNA in COVCAR cells. Total RNA extracted from COVCAR cells (n = 5 cell lines; C5, C6, C7, C11, and C19) and normal ovarian surface epithelial cell lines (NOSE; n = 5 cell lines), was DNAse-I digested and reverse transcribed. Following reverse transcription, approximately 50 ng of cDNA was used in quantitative real-time PCR using SYBR® green as the dye to quantify AQP5 mRNA and β-actin mRNA in separate reactions. Each reaction was run in triplicate per cell line; and the critical threshold (CT) values, averaged, normalized to that of β-actin mRNA and converted from log-linear to linear term; B. Quantification of AQP5 protein in COVCAR cells. Protein extracts from COVCAR cells (n = 5 cell lines; C5, C6, C7, C11 and C19), and NOSE cells (n = 5 cell lines) were subjected to Western blot analysis under reducing conditions. Chicken AQP5 protein was detected by immunostaining using anti-human AQP5 antibody and chemiluminescence. Alpha-tubulin was used as loading control. Data are represented as mean ± standard error of the mean from 5 replicates. *P < 0.05; **P < 0.001 COVCAR versus NOSE cells.