Fig. 1.
Validation of SLC26A6 silencing using small interfering RNA (siRNA) techniques in Caco-2 monolayers. A: development of transepithelial resistance (RT) is presented for Con and A6KD monolayers seeded at 4 × 105 cells/cm2. By the fourth day postseeding there were no significant differences in RT measured between control (Con) and SLC26A6 knockdown (A6KD) monolayers (n = 9). B: persistence of siRNA-induced depression of SLC26A6 mRNA. The expression of SLC26A6 mRNA relative to GAPDH mRNA from Con, scrambled siRNA (Scram), and A6KD monolayers were quantitated by real-time RT-PCR (n = 6). The relative change in expression for Scram and A6KD were computed by the ΔΔCT method as provided in the Relative Expression Software Tool spreadsheet [REST384 (20)] to statistically evaluate the fold changes in gene expression. The numbers above each bar are the probability values generated by the REST analysis for a comparison of a sample with its respective controls. Scrambled siRNA did not significantly affect the expression of SLC26A6 mRNA, whereas siRNA for SLC26A6 produced significant reduction in SLC26A6 message at days 2 and 6. C: relative abundance of SLC26A6 to GAPDH in Con, Scram, and A6KD monolayers 6 days postseeding. Error bars represent ±1 SE (n = 4); *A6KD monolayers are significantly different from Con and Scram (in a 1-way ANOVA). Inset shows an immunoblot for SLC26A6 and the reference protein GAPDH in the same membrane.