7500 step oneplus real time pcr system

RNA was isolated using an RNeasy kit (Qiagen) and reverse transcription was performed using SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). Quantitative RT-PCR was performed using SYBR® Green PCR Master Mix (Applied Biosystems™) on the 7500 Step OnePlus™ Real-Time PCR System (Applied Biosystems^TM). All samples were run in triplicate and relative quantification was done using the ΔΔCt method with normalization to GAPDH. A list of primers can be found in Supplementary Table 2. Genomic DNA was isolated using a DNeasy kit (Qiagen) and sequencing was performed by LGC Genomics (Teddington, UK). The TaqMan® hPSC Scorecard™ Panel (Life Technologies—A15870—HPSC scorecard panel 384) kit was used to determine the expression of markers from the three germ layers.

Total RNA extraction was performed with the RNeasy Mini kit (Qiagen). cDNA was synthesized from 1 μg of total RNA using SuperScript III™ First-Strand Synthesis SuperMix (Invitrogen), according to the manufacturer’s instructions. Quantitative PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Waltham, US) on the 7500 Step OnePlus Real-Time PCR system (Applied Biosystems). Relative gene expression was determined by the 2^−ΔΔct method and normalized to ZNF48 transcript levels. The following primers (IDT) were used: HNRNPK (forward 5ʹ-GGTGGCTCCGGATATGATTATT-3ʹ, reverse 5ʹ-TGGTCCTGTGTTCCTGTAATG-3ʹ), ZNF48 (forward 5ʹ-CAAACACCAGCGGACTCATA-3ʹ, reverse 5ʹ-TGCCACATTCACCACAGATAG-3ʹ).

Total RNA extraction was performed with the GenElute Mammalian Total RNA Kit (Sigma-Aldrich). cDNA was synthesized from 1 μg of total RNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen) according to the manufacturer’s instructions. The qPCR for Sendai virus detection was performed using TaqMan Gene Expression Assays (Life Technologies). Quantitative RT-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) on the 7500 Step OnePlus Real-Time PCR System (Applied Biosystems). Relative gene expression was determined by the 2^−ΔΔCt method with normalization to GAPDH mRNA.