RNA extraction and quantitative real-time polymerase chain reaction

Total RNA was isolated with TRIzol reagent (15596, Invitrogen) and cDNA was synthesized from 2 μg of total RNA with oligo-dT using Superscript III cDNA synthesis kit (18080-093, Invitrogen) for 30 min at 50 °C, followed by 2 min at 94 °C to inactivate the reverse transcriptase. The expression levels of Oct4, Nanog, Sox2 and GAPDH in all sample were assessed by real-time PCR using SYBR Green (04913914001, Roche) with following cycling conditions: 95 °C for 10 min, 40 cycles of 95 °C for 15 sec, 60 °C for 1 min, and 72 °C for 20 sec. Standard curves (cycle threshold values versus template concentration) were prepared for each target gene and for the endogenous reference [glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] in each sample. Quantification of unknown samples was performed using LightCycler Relative Quantification Software version 3.3. The following primers sequences were used: GAPDH ({"type":"entrez-nucleotide","attrs":{"text":"NM_001289745.1","term_id":"576583518","term_text":"NM_001289745.1"}}NM_001289745.1) Forward primer 5′-CTCTGCTCCTCCTGTTGTTCGACA-3′; Reverse primer 5′-ACGACCAAATCCGTTGACTC-3′; Sox2 ({"type":"entrez-nucleotide","attrs":{"text":"NM_003106","term_id":"1519313016","term_text":"NM_003106"}}NM_003106) Forward primer 5′-ATGCACCGCTACGACGTCA-3′; Reverse primer 5′-CTT TTGCACCCCTCCCAT TTT-3′; Nanog ({"type":"entrez-nucleotide","attrs":{"text":"XM_011520852.1","term_id":"767972407","term_text":"XM_011520852.1"}}XM_011520852.1) Forward primer 5′-ATGCCTGTGTTTGTGGGCC-3′; Reverse primer 5′-GCCAGTTGTTTTTCTGCCAC-3′; Oct4 ({"type":"entrez-nucleotide","attrs":{"text":"NM_001159542.1","term_id":"227430409","term_text":"NM_001159542.1"}}NM_001159542.1) Forward primer 5′-AGCCCTCATTTCACCAGGCC-3′; Reverse primer 5′-CAA AACCCGGAGGAGTCCCA-3′.