Mastercycler ep realplex es

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.

Total cellular RNA was extracted with Trizol (Invitrogen) [19 (link)] and subjected to DNase treatment (Ambion). Amounts were normalized against ribosomal RNA in each sample. cDNAs were synthesized from 2 µg of total RNA using the RETROscript first strand synthesis kit (Ambion). A real-time thermocycler (Eppendorf Mastercycler ep realplex ES) was used to perform qRT-PCR. SYBR Green fluorescence was measured, and relative quantification was made against the RPS9 cDNA used as an internal standard. Gene-specific primers for qRT-PCR (rat InsI for GACCCGCAAGTGCCACAA, rev TCCACAAGCCACGCTTCTG; human OCN for TGACGAGTTGGCTGACCA, rev AGGGTGCCTGGAGAGGAG) were designed according to sequences from the GenBank database.