Differences in the Gene Expression Profiles of Slow- and Fast-Forming Preinduced Pluripotent Stem Cell Colonies

Figure 1

Multiplex reverse transcription-polymerase chain reaction for pluripotency genes in NGFP and 4F2A cells. (a) A schedule for doxycycline (Dox) induction in NGFP-mEF and 4F2A-mEF culture. Initially formed-iPSC (if-iPSC) colonies of proper sizes were picked at indicated times. Dox treatment was maintained until colony picking. (b) The number of pre-iPSC colonies emerged from NGFP-mEF (272 29 per 2 10⁴ cells) and 4F2A-mEF (188 24). Mean standard deviation. . (c) RT-PCR of individual genes using cDNA from mouse J1 mESCs. The size of PCR amplicon is indicated below. (d) Multiplex RT-PCR strategy. One-step RT-PCR was used as the first round of PCR (PCR-1) and the products advanced to the second-round, nested PCR (PCR-2). (e) A representative gel image (upper) and the corresponding band-intensity profiles of the groups 1 and 2 genes (lower). (f) Control experiments for multiplex RT-PCR using total RNA from J1 mESCs. Omitting each set of primers indicated (a–d) in the PCR-1 cocktail does not greatly change the overall pattern and quantity of the PCR-2 products of the remaining genes in each group, indicating no severe interference between the multiple sets of primers during multiplex RT-PCR.