Phenol free and filter based rna isolation system

PCR and qRT-PCR were performed as previously described (12 (link)). Briefly, a PCR kit (Qiagen, Valencia, CA) was used to genotype the SALL4B Tg founder mice and transmission of the transgene. Genomic DNA was purified from a mouse tail using a high-quality DNA kit (Gentra Systems, Minneapolis, MN). The human SALL4B primer sequences were showed in supplementary table 1. Total RNA was isolated using a phenol-free and filter-based RNA isolation system (Qiagen) digested with DNase I to remove DNA contamination. Primer sequences for qRT-PCR were designed using Primer Express® software (Applied Biosystems, Foster City, CA) and were listed in supplementary table 1. All reactions were performed in an ABI-7000 sequence detection system using TaqMan PCR core reagents according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). PCR amplification was carried out in a 50 μl final volume containing 1×PCR buffer, 1 μM of each primer, and 10 μl of RNA at 95°C for 10 min followed by 45 cycles at 95°C for 15 sec and then at 60°C for 1 min. For each sample, expression of the GAPDH gene was used to normalize the amount of investigated transcript.