All the Tested Human Somatic Cells Express Both Oct4A and Its Pseudogenes but Express Oct4A at Much Lower Levels Compared with Its Pseudogenes and Human Embryonic Stem Cells.

Oct4 pseudogenes and isoforms seriously confuse the detection of the pluripotency-associated Oct4A expression in somatic cells, which in many cases was not accurately determined. This confusion has recently been discussed, but the wrong conclusions have continuously been made. Most studies failed to detect the expression of Oct4 pseudogenes and isoforms in somatic cells but detected only Oct4A, for which the detection signals incorrectly came from its pseudogenes and isoforms. Some studies detected the expression of only Oct4 pseudogenes in somatic cells but failed to detect Oct4A. The other studies failed to detect the expression of any Oct4 genes. Oct4A is more homologous to its pseudogenes than its isoforms, and it is much more difficult to distinguish Oct4A from its pseudogenes, so this study focused on them. In this study, the strict experimental procedures were followed. Three pairs of Oct4A-specific polymerase chain reaction (PCR) primers were carefully designed and tested by sequencing reverse transcription-polymerase chain reaction (RT-PCR) clones, which showed that only one of them was truly specific to Oct4A. RT-PCR was also performed with the primers amplifying both Oct4A and its pseudogenes, and several hundreds of PCR clones from each cell type were sequenced to reliably distinguish the low-abundant Oct4A from its high-abundant pseudogenes. Western blot, immunocytochemistry, and flow cytometric analyses were performed with three Oct4 antibodies to confirm the results of Oct4 mRNA expression. This study undoubtedly made the correct conclusions about Oct4 expression in human somatic cells and showed that all the tested human somatic cells expressed both Oct4A and its pseudogenes but expressed Oct4A at much lower levels compared with its pseudogenes.