Journal: Frontiers in Genetics

Article Title: Simultaneous Methylation-Level Assessment of Hundreds of CpG Sites by Targeted Bisulfite PCR Sequencing (TBPseq)

doi: 10.3389/fgene.2017.00097

Figure Lengend Snippet: Validation of the LIFR promoter methylation for cancer specificity and its relationship with the expression of associated genes. (A) DNA methylation levels of target CpGs in public cancer methylome data (Infinium 450K BeadChip array) of four cancer types: colorectal ( n = 313 for cancer samples and n = 38 for normal samples), liver (377 and 50), lung (843 and 74), and stomach (395 and 2) cancers. Infinium CpG identification numbers (IDs) together with the associated gene names are shown; the Infinium IDs cg03723506 and cg11291081 indicate chr5:38557143 and chr3:37033894, respectively, in the Figure . Statistical significance was calculated using Wilcoxon rank sum test. T: tumor samples, N: normal samples. (B) Schematic drawing of COBRA region at the LIFR promoter. Blue arrows, primers. CGI, CpG island (green line). (C) COBRA analysis. Genomic DNA was extracted from each colon cancer cell lines along with a normal control colon cell line (CCD-18co) and subjected to COBRA using the Taq I enzyme to examine the methylation state at the LIFR gene promoter. Arrowhead and arrow indicate the positions of intact and Taq I-digested DNA fragments, respectively. The fraction (% meth) of methylated DNA was measured by band intensity analysis and noted under each cell line. (D) RT-PCR. The same cancer cell lines used in COBRA (C) were subjected to RT-PCR to measure the transcript levels of the LIFR and LIFR-AS genes.

Article Snippet: The panel composition is highly flexible and can accommodate a variety of experimental designs, a big advantage over other methylation analysis platforms such as the array-based Infinium BeadChip.

Techniques: Biomarker Discovery, Methylation, Expressing, DNA Methylation Assay, Combined Bisulfite Restriction Analysis Assay, Control, Reverse Transcription Polymerase Chain Reaction