Expression of endogenous (canine) mRNA and Pgp protein in MDCK wildtype cells is high, whereas expression of endogenous (porcine) mRNA and Pgp protein in LLC cells is low or undetectable. instructions. The concentrations and purity of RNA were estimated spectrophotometrically at 260 and 280 nm, and the integrity of the RNA was checked by electrophoresis on 1% agarose gels. RNA was reverse-transcribed with oligo(dT)20 and random hexamer primers in a final volume of 10 L using SuperScript III First-Strand cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Gene expression of (human), (dog), (pig) and dog and was evaluated by qPCR. Amplification was detected by SYBR? Green fluorescence on a Mx3005P instrument (Stratagene, La Jolla, CA, USA). cDNA of each sample was diluted 1:24 in RNase-free water. Thermocycling was carried out in a final volume of 20 L containing 10 L Precision?-MX-SY MasterMix 2x (PrimerDesign Ltd, Southampton, UK), 1 L of each forward and reverse primer (final concentration 0.5 M) and 8 L diluted cDNA. The thermal cycling conditions were 10 min at 95C to activate DNA polymerase, followed by 45 amplification cycles at 94C for 10 s, 58C for 10 s and 72C for 30 s, with the last cycle finished by 3 min elongation at 72C and dissociation curve analysis to ensure the specificity of the PCR product. As negative controls, we used = 0), the pre-incubation medium was replaced by fresh Opti-MEM? containing the drug in both chambers (see below). The volumes in the upper and lower compartment were 2000 L and 2700 L respectively. For drug analysis, samples were Timosaponin b-II taken at 60, 120, 240 and 360 min. The transport assays including pre-incubation were performed at 37C in a humidified incubator (5% CO2) with shaking the transwells gently at 50 rpm. Monolayers were checked for integrity by measuring transepithelial electrical resistance (TEER) of the polarized cells before and after each transport experiment and by using [14C]-mannitol (in separate wells) as described recently (Luna-Torts 0.05 was considered significant. Results Expression of endogenous (canine) mRNA was strikingly lower in mRNA was determined in MDCK wildtype cells (Figure 1B), thus confirming the specificity of the primers. The expression of human mRNA in the Timosaponin b-II transfected cells was about threefold higher than the expression of endogenous canine mRNA in wildtype cells (Figure 1A,B). When the Pgp protein content was determined in MDCK and MDCK-cells by Western blot, total (endogenous canine and recombinant human) Pgp expression in the transfected cells was about three times higher than endogenous Pgp expression in the wildtype cells (Figure 1C). Open in a separate window Ly6c Figure 1 Expression of endogenous (canine) in parental (wildtype; WT) and is about seven times lower in mRNA in the transfected cells and the lack of any human expression in wildtype cells. C illustrates the protein content of Pgp in MDCK wildtype and 0.001. Data in (D) illustrate a representative experiment in which qPCR was used to determine mRNAs of canine and in MDCK wildtype cells versus cells transfected with human and mRNAs in the transfected cells. MDCK, Madin-Darby canine kidney; MDR1, multidrug resistance 1; Pgp, P-glycoprotein; qPCR, quantitative PCR. In addition to examining whether the endogenous mRNA expression differs between transfected and wildtype cells, we also determined the expression Timosaponin b-II of endogenous Mrps in MDCK.