Therefore, to identity of the Oct-3/4 RT-PCR product from the neural stem cells, we performed nested PCR followed by DNA sequencing. The PCR reaction was performed using the first PCR primer set and the first PCR product was used as a template for the second PCR reaction. The primer set 1 (PS1), which has been used in the previous study [12] encompasses the
apexbio 1 of Oct-3/4 gene (Fig. 1). The PCR reaction with primers of PS1 could possibly amplify the pseudogenes on chromosome 3 (XR_034544.1 and XR_034265), but not the pseudogene on chromosome X (NW_001035169.1) based on the sequence similarities of primers to the pseudogenes. We used cDNA from mouse embryonal teratocarcinoma
cells line, P19, as a positive control for Oct-3/4 PCR. The first PCR only amplified the expected band from the cDNA of P19 cells but not neural stem cells (NSCs). However, the second PCR was able to amplify a band with expected size (268 bp) from NSCs, while no products were amplified in minus reverse transcriptase control (Fig. 2A). To further confirm, we designed an additional primer set (PS2) that encompasses the exon 1 through the exon 4 (Fig. 1) so that genomic DNA can be discriminated. As observed in the PCR with PS1, the second PCR with PS2 amplified a product with expected size (472 bp) from NSCs with no products in the minus reverse transcriptase control (Fig. 2B).
