Fig. 6

NOS2 mRNA isoform analysis using 5′RACE and isoform specific qRT-PCR methods. A Schematic summary of the results of 5′-RACE analyses using RNA isolated at day 18 from human iPSC induced to differentiate to neurons. RNA was isolated from 4 different human iPSC lines (samples C16-1, C16-2, C89-2, C89-3) induced to differentiate to neurons [53] for 18 days. As control RNA was isolated from human DLD1 cells (DLD1-2 and DLD1-2) treated with a cytokine mixture for 6 h to induce NOS2 mRNA expression. Then 5′-RACE experiments were performed (as described in “Methods” section) using the indicated oligonucleotides as 3′-primer (5′RACE-rev = primer used for the RT-reaction; 3P2/3P3 primers used for the PCR-reaction). The final PCR products were isolated, cloned into pCR-Script and sequenced. The alignment of these sequences to the human NOS2-1 and -2 mRNA are indicated as filled arrows. The sequences of the fragments are shown in Additional file 1: Fig. S10. B/C NOS2-mRNA-isoform expression analysis using isoform specific qRT-PCR experiments. RNA was isolated at different time points from 2 different human iPSC lines (iLB-C16bm s6 iPS andiLB-C89bf s4 iPCs) induced to differentiate to neurons [53] for 60 days. The RT reaction was performed with the RT-rev primer (see A). Taqman qPCR reactions were performed with mRNA isoform specific primer pairs and taqman probes (NOS2-1: NOS2-1_P, NOS2-1_3P and NOS2-1_Taq; NOS2-2: NOS2-2_5P, NOS2-2_3P and NOS2-2_Taq—see A). For normalization also the GAPDH mRNA expression was measured. B The normalized NOS2-1 mRNA expression values were related to the CM induced NOS2-1 mRNA expression in DLD-1 cells (CM = 100%). Also, the expression level of the NOS2-1 mRNA in untreated (co) DLD-1 cells were determined. (***p < 0.001, ns not significant vs. iPSC treated for 18 days; 1-way Anova with Dunnett’s multiple comparisons test). C The normalized NOS2-2 mRNA expression values were related to the NOS2-2 mRNA expression in iPSC treated for 18 days (day 18 = 100%). (**p < 0.05, vs. iPSC treated for 18 days; 1-way Anova with Dunnett’s multiple comparisons test)