2.4 RNA extraction and real-time PCR analyses
Following manufacturing’s guideline, RNA was extracted employing RNAiso Plus (TaKaRa Cat# 9109, Kusatsu, Shiga, Japan). The isolated total RNA is then converted into cDNA by means of the Primescript RT Reagent Kit (TaKaRa Cat# RR037A, Kusatsu, Shiga, Japan) following the manufacturer’s practice. The cDNA was next utilized for quantitative PCR analysis by an ABI Prism 7300 Real‐Time PCR System (Applied Biosystems, Foster City, CA, USA) via qPCR Master Mix (Bimake Cat# B21203, Houston, TX, USA). Quantification of mRNA levels of human and mouse genes i.e., Axin2, LEF1, Fibronectin, Survivin, OCT4, Twist1, LGR5, and SOX2 were carried out. The “comparative Ct method” was accomplished to evaluate relative countenance of genes. The data are obtained as the fold change. The fold change was premeditated as 2-ΔΔCt (ΔΔCt =ΔCttreated - ΔCtcontrol., Ct is the cycle figure at spot where fluorescence first surpasses the threshold). The ΔCt values from each target gene were obtained by subtracting the values for GAPDH Ct from the illustration Ct. The data of three technical replicates/sample are presented. The primer sequences are shown in supplementary tables (Supplementary Table S1 and S2).