Figure 1
From: Epigenetic silencing of monoallelically methylated miRNA loci in precancerous colorectal lesions
Identification of the pri-miR-497/195 TSS in normal human colorectal mucosa and assessment of the methylation status of the CpG island located upstream from this site. (a) 5′ RACE: a 3638-bp product was obtained by nested PCR with the gene-specific outer and inner primers listed in Material and methods. (b) Schematic showing locations of miR-497 and miR-195, the pri-miR-497/195 TSS and the CpG island located upstream from the latter (genome coordinates of pri-miR-497/195 from its TSS to miR-497 according to the Human Reference Sequence GRCh37/hg19: chr17, 6 921 342–6 924 948). The position of the 294-bp COBRA amplicon is 6 926 487–6 926 780. (c) COBRA analysis of six colorectal cancer cell lines and a control sample of normal colon mucosa. Arrows indicate TaqαI-digested DNA fragments representing methylated alleles; slower-migrating fragments represent undigested, unmethylated DNA. (d) COBRA revealed methylated and unmethylated pri-miR-497/195 alleles in isolated epithelial cells and isolated stromal (lamina propria) cells from the normal mucosa. (e) Left panel: the purity of the epithelial cell preparation was confirmed by qPCR exclusion of the expression of Vimentin, a stromal marker that was abundant in the lamina propria specimens. Right panel: RT–PCR revealed higher expression of miR-195 and miR-497 in the stromal component.
