Quantitative Transcriptional Analysis of Diverse Cell Types

Total RNA was extract from human fibroblast (C-013-5C, Life Technologies), human iPS cells (SBI) and hDSCs by the single-step method of isolation using Trizol (Invitrogen), and then was reverse transcribed with random hexanucleotides using the SuperScript III First-Strand Synthesis System (Invitrogen) as prescribed described69 (link). PCR was performed with JumpStart Taq DNA polymerase (Sigma). In quantitative PCR, 50 ng of total RNA was typically used as template in 20 ml SYBR green PCR reactions (40 cycles of 15 s, 95 °C/60 s, 60 °C) on Applied Biosystems 7300 that additionally contained 0.375 mM of each primer70 (link)71 (link) and 10 ml of SYBR green PCR mix (ABI).