Sybr green realmastermix

For qRT-PCR, total cellular RNA was extracted from cells using the RNAqueous-4PCR kit and subjected to DNase treatment (Ambion). RNA levels were normalized against 18S ribosomal RNA in each sample, and cDNAs were synthesized from 2 μg of total RNA using the RETROscript first strand synthesis kit (Ambion). Primers were designed according to sequences from the GenBank database: human IGFBP1 (NM_000596.2): for 5′-CATTCCATCCTTTGGGAC-3′; rev 5′-ATTCTTGTTGCAGTTTGGCAG-3′. human INSR (NM_000208.2) for 5′-TTTGGGAAATCACCAGCTTGGCAGAAC-3′; rev. 5′-AAAGCTGGGGTGCAGGTC GTCCTTG-3′. A real-time thermocycler (Eppendorf Mastercycler ep realplex ES) was used to perform quantitative PCR. In a 20 μL final volume, 0.5 μL of the cDNA solution was mixed with SYBR Green RealMasterMix (Eppendorf), and 0.3 μM each of sense and antisense primers. The mixture was used as a template for the amplification by the following protocol: a denaturing step at 95°C for 2 min, then an amplification and quantification program repeated for 45 cycles of 95°C for 15 s, 55°C for 25 s, and 68°C for 25 s, followed by the melting curve step. SYBR Green fluorescence was measured, and relative quantification was made against ribosomal protein S9 cDNA used as an internal standard.

Cells were serum-starved overnight and inhibitors or solvent alone were applied one h prior to EGF-stimulation. Cells were stimulated with 25 ng/ml EGF or left unstimulated, for four h prior to harvesting.
Total RNA was extracted using RNeasy kit (QIAGEN, USA), and complementary DNA (cDNA) was synthesized by RT-RTCK-05 kit (Eurogentec, Berlin, Germany) and stored at −20 °C. A standard real-time PCR reaction with SYBR green Real MasterMix (Eppendorf, Hamburg, Germany) was performed in duplicates using Mx3005p (Agilent Technologies, USA) under the following conditions: 95 °C for 2 min followed by 40 cycles of 95 °C for 20 s, 60 °C for 1 min and 68 °C for 30 s. Dissociation curves ensured product uniformity. Expression data was normalized to the housekeeping gene TATA-box binding protein (TBP). The relative expression levels of the gene of interest were calculated using the 2^-ΔΔCt method. AREG primers were obtained from Sigma-Aldrich: forward 5′-GCT-CAG-GCC-ATT-ATG-CTG-CTG-3′, reverse 5′-ACT-CAC-AGG-GGA-AAT-CTC-ACT-CC-3′; TBP primers were obtained from Eurogentec: forward 5′-CGT-GGC-TCT-CTT-ATC-CTC-ATG-A-3’, reverse 5’-GCC-CGA-AAC-GCC-GAA-TAT-A-3’.