Gene Expression Profiling of Intestinal Cell Lines

The mRNA expression levels of the Lgr5, Mki67, Muc2, Vil1, Chga, Lyz, Cdh1, and Hprt genes in 2D-hiPSC-SI were measured by real-time reverse transcription PCR (RT-PCR). Hprt was used as an endogenous control for data standardization. Total RNA was isolated from 2D-hiPSC-SI using an Agencourt RNAdvance Tissue kit (Beckman Coulter Inc., Brea, CA, USA). In order to create single-stranded cDNAs, 0.5 µg of total RNA was used. ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan) was used for the reverse transcription process in accordance with the manufacturer’s instructions. Real-time PCR analysis was carried out on an Eco Real-Time PCR system using Eco Real-Time PCR System software, version 5.0 (Illumina Inc., San Diego, CA, USA). PCR was performed with the primer pairs indicated in Table S1 using KAPA SYBR FAST qPCR Kit Master mix (2×) and an ABI Prism system (Sigma-Aldrich, St. Louis, MO, USA) [26 (link)].