Biomark real time pcr analysis software 4

Total RNA was extracted from hiPSCs and differentiated cells using the PureLink^® RNA Mini Kit (Life Technologies) according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of RNA per sample, using High-Capacity RNA-to-cDNA™ Kit (Life Technologies). cDNA was diluted 10-fold in DNA suspension buffer (Teknova, Inc, Hollister, CA, USA) and used for the Fluidigm pre-amplification step for Dynamic Array gene expression analysis: cDNA was pre-amplified for 14 cycles with 500 nM DELTA gene pooled primer mix using 2xTaqman PreAmp Master Mix (Invitrogen, Carlsbad, CA, USA), followed by Exo1 treatment (NEB). Five-fold diluted Exo1 treated pre-amplified cDNA was used for loading the 96.96 Dynamic Array chip on the Fluidigm Biomark HD. Primer pairs used for gene expression analysis are listed in the supplemental information (Table S1). The data were analyzed with Real-Time PCR Analysis Software in the BioMark instrument. The Ct values were processed by the automatic threshold for all assays, with derivative baseline correction using BioMark Real-Time PCR Analysis Software 4.1.3 (Fluidigm, South San Francisco, CA, USA).

Total RNA was extracted from hiPSCs and differentiated cells using the PureLink^® RNA Mini Kit (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instructions. cDNA was synthesized from 1 μg of RNA per sample, using High-Capacity RNA-to-cDNA™ Kit (Life Technologies, Carlsbad, CA, USA). cDNA was diluted 10-fold in DNA suspension buffer (Teknova) and used for the Fluidigm pre-amplification step for Dynamic Array gene expression analysis: cDNA was pre-amplified for 14 cycles with 500 nM DELTAgene pooled primer mix using 2x Taqman PreAmp Master Mix (Invitrogen), followed by Exo1 treatment (NEB). Five-fold diluted Exo1 treated pre-amplified cDNA was used for loading the 96.96 Dynamic Array chip on the Fluidigm Biomark HD. Primer pairs used for gene expression analysis are listed in Supplementary Table S1. The data were analyzed with Real-Time PCR Analysis Software in the BioMark instrument. Ct values were processed by automatic threshold for all assays, with derivative baseline correction using BioMark Real-Time PCR analysis Software 4.1.3 (Fluidigm). Statistical significance for relative fold change values was determined using Student’s t-test.