Applied biosystems 7500 real time polymerase chain reaction system

We investigated the gene expression profile of the transcription factor NeuroD1 and the endocrine hormone insulin as we reported previously.^{18 (link)} Total RNA was extracted from cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and complementary DNA was synthesized from 2.5 μg of total RNA by reverse transcription using a SuperScript RT kit (Promega, Madison, Wis), following the manufacturer's instructions. Then, quantitative reverse transcription—polymerase chain reaction was performed using the Applied Biosystems 7500 real-time polymerase chain reaction system, TaqMan Gene Expression Assays on demand, and a TaqMan Universal Master Mix (gene-specific TaqMan probes on a StepOne Plus; Applied Biosystems, Foster City, Calif). NeuroD1 (NM_019218) and Insulin1 (NM_019129) primers (Qiagen) were used. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control for normalization. Expression levels of all genes were calculated as a ratio to glyceraldehyde-3-phosphate dehydrogenase. Amplification data were analyzed with an Applied Biosystems Prism 7500 Sequence Detection System version 1.3.1 (Thermo Fisher Scientific Inc).