To investigate the relationship between RSPO3 promoter methylation and its mRNA expression, we examined RSPO3 expression and its promoter methylation by bisulfite sequencing PCR (BSP) of genomic DNA from CCA cells (TFK1, EGI1, QBC939, HuCCT1, HuH28, RBE, HCCC9810, and SSP25) after bisulfite processing (Fig. 2A). This evidence concerns the gene RSPO3 and cholangiocarcinoma.