Pcr mastercycler ep gradient

The RPE characteristics of hiPSC-RPEs was analyzed with RT-PCR using complementary DNA as a template. The reaction was done using 5 μM primers specific for specific genes (Biomers.net GmbH, Söflinger, Germany, Table 1), 5 U/μL Taq DNA Polymerase (Fermentas, Thermo Fisher Scientific Inc., Leicestershire, U.K.) in PCR MasterCycler ep gradient (Eppendorf AG, Hamburg, Germany) according the protocol: 95 °C 3 min, 95 °C 30 s, annealing 30 s, 72 °C 1 min, 72 °C 5 min, for 38 cycles. Annealing temperatures and primer sequences are presented in Table 1. PCR products were resolved in 2% agarose gels with a 50-bp DNA ladder (MassRulerTM DNA Ladder Mix, Fermentas, Thermo Fisher Scientific Inc., Leicestershire, U.K.). The products were visualized with the Quantity One 4.5.2. Basic program (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

In order to analyze the gene expression of mature hESC-RPE cells, total RNA was extracted from hESC-RPE cells with a NucleoSpin XS-kit (Macherey-Nagel, GmbH & Co., Düren, Germany) according to the manufacturer’s instructions. The RNA concentration and its quality were assessed using NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA (40 ng) was reverse-transcribed to complementary DNA using MultiScribe Reverse Transcriptase (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions in the presence of an RNase inhibitor. Complementary DNA (cDNA) was used as a template in a PCR reaction, which was carried out using 5 U/μL Taq DNA Polymerase (Fermentas, Thermo Fisher Scientific Inc., Leicester, UK) with 5 μM specific primers (Biomers.net GmbH, Söflinger, Ulm, Germany, Table 3). The PCR reactions were carried out in PCR MasterCycler ep gradient (Eppendorf AG, Hamburg, Germany) as follows: 95 °C 3 min, 95 °C 30 s, annealing 30 s, 72 °C 1 min, 72 °C 5 min, for 38 cycles. Annealing temperatures and primer sequences are presented in Table 3 and Figure S1. PCR products were resolved in 2% agarose gels with a 50-base pair DNA ladder (MassRulerTM DNA Ladder Mix, Fermentas). The bands were visualized with the Quantity one 4.5.2. Basic program (Bio-Rad Laboratories, Inc., Hercules, CA, USA).