7900ht fast real time pcr system cycler

Gene expression analyses were carried out on Caco-2 cells to determine the direct action of treatments on gene expression of SGLT1 and GLUT2 glucose transporters. Caco-2 cells seeded at 2 × 10⁵ cells/well in a 6-well plate were incubated for 48 h treated with either 100 μM pure peptides (AKSPLF, ATNPLF, FEELN, LSVSVL), or 10 mg/mL PFRA or 100 μM PHL. After pre-treatments for two h on glucose free media, cells were stimulated with 20 mM glucose during 30 and 24 h. Then, RNA was collected using a RNAeasy kit (Qiagen, Germantown, MD), quantified, and checked for quality. cDNA was synthesized using a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City CA). Quantitative q-PCR was performed using TaqMan® gene expression assay in a 7900HT Fast Real-Time PCR System Cycler (Applied Biosystems, Foster City, CA) and targeted human genes SGLT1 (SLC5A1), GLUT2 (SLC2A2), GAPDH (Gapdh2). Thermocycling conditions were: one cycle at 95 °C for 10 min and then 40 cycles at 95 °C for 15 s followed by 60 °C for one min before a dissociation stage. The relative mRNA expression levels of each gene were calculated using the 2^−ΔΔCt method in reference to GAPDH.