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).