Oligo dt 12 18 primer

Total RNA purified from ES and TS cells was reverse transcribed using Superscript II (TaKaRa, Otsu, Japan) and an oligo(dT)12–18 primer (TaKaRa) in a total volume of 20 μl. Quantitative real-time RT-PCR was performed using SYBR Premix Ex Taq (Perfect Real Time, TaKaRa). The PCR mixture consisted of 2 × SYBR Premix Ex Taq, 10 μM forward and reverse primers, and template cDNA in a total volume of 12.5 μl. The cocktail was activated by heating at 95°C for 10 sec. The subsequent PCR reaction was carried out at 95°C for 5 sec and 60°C for 30 sec for 40 cycles in a LightCycler480 (Roche). PCR amplification was performed using the following primer sets:
p53: 5′-GTCACGCTTCTCCGAAGACT-3′
and 5′-GTCCATGCAGTGAGGTGATG-3′
Bax: 5′-GCTGGACACTGGACTTCCTC-3′
and 5′-GAGGACTCCAGCCACAAAGA-3′
Bcl-2: 5′-AGTACCTGAACCGGCATCTG-3′
and 5′-GCTGAGCAGGGTCTTCAGAG-3′
Oct4: 5′-CCAATCAGCTTGGGCTAGAG-3′
and 5′-CTGGGAAAGGTGTCCCTGTA-3′
Nanog: 5′- ATGCCTGCAGTTTTTCATCC-3′
and 5′- GAGGCAGGTCTTCAGAGGAA-3′
Sox2: 5′- GAGTGGAAACTTTTGTCCGAGA-3′
and 5′- GAAGCGTGTACTTATCCTTCTTCAT-3′
Stella: 5′- AGCCGTACCTGTGGAGAACAA-3′
and 5′-TCTTTCAGCACCGACAACAAA-3′
Fgf5: 5′-CTCAGGGGATTGTAGGAATACGAGGA-3′
and 5′-GGATCGCGGACGCATAGGTATTATAG-3′
Gapdh: 5′-AATGCATCCTGCACCACCAA-3′
and 5′-GTGGCAGTGATGGCATGGAC-3′
The Gapdh gene was used to standardise the data.

Total RNA was extracted from cultured cells using TRIzol^® reagent (Invitrogen, NY). Single-stranded cDNA was generated from total RNA, using M-MLV reverse transcriptase and oligo (dT) 12–18 primers (Takara, Japan). The real-time quantitative PCR reaction was performed with the SYBR green detection system (Bio SYBR Green Master Mix, Takara, Japan). GAPDH served as an endogenous control. All cycle threshold (ct) values were determined in real time using CFX96™ Real-Time PCR Detection (Bio-rad, CA). The primer pairs for each target gene is listed in Supplementary Table 2.