Gene expression analysis
Expression of 24 genes was evaluated by qRT-PCR (AFP, ALB, CK18,
CK19, CXCR4, CYP3A4, FOXA2, G6PC, HNF1A, HNF4A, NANOG, POU5F1, SOX2,
SOX17, TDO2, UGTA1, TET2, TET3, DNMT1, DNMT3A, DNMT3L, SOX2, TET1, andUHRF1 - primers upon request). The cDNA was synthesized using the
High Capacity RNA-to-cDNA kit (Applied Biosystems, EUA). Experiments
were based on the International Guideline MIQE (The Minimum Information
for Publication of Quantitative Real-Time PCR Experiments). The
housekeeping genes were chosen using the geNorm algorithm20 after expression analysis of ACTB ,GAPDH , B2M genes and 18S ribosomal RNA (18S rRNA ).
Data were normalized using the expression values of the housekeeping
genes 18S rRNA and B2M , and all reactions were performed
with three technical replicates. The delta-delta Ct (∆∆Ct) method was
used for data analysis 21.