MethylScreen technology principles and data interpretation.

<p>(A) Representative qPCR standard curve for PAX6 obtained from 100, 50, 10, 1 and 0.1 ng of control DNA per reaction. (B₁) <i>UTF1</i> MethylScreen qPCR results for CCTL-12, IPSCF, CBIA-11, A549 and KG-1 genomes. Changes in c_t between enzyme treated and non-treated templates are depicted: Rs-R0 is represented by white columns (HhaI reaction), Rd-R0 is represented by grey columns (McrBC reaction), and Rsd-R0 is represented by black columns (both HhaI and McrBC reaction). (B₂) <i>UTF1</i> DNA methylation profile for five cell lines. c_t values from four restriction reactions (B₁) were converted to DNA methylation occupancy, expressed as the percentage of unmethylated (UM), intermediately methylated (IM) and hypermethylated (HM) DNA. (C) MethylScreen results obtained from sample mixtures with an increasing ratio of hypermethylated DNA (0–100%, x-axis). The colour legend is identical for (B₂), and the values are averages for <i>PAX6</i> and <i>TSPYL5</i> genes after serial mixing of CCTL-14 (unmethylated) and A549 (hypermethylated) samples and NDFs (unmethylated) and CCTL-14 (hypermethylated) samples, respectively.</p

DNA methylation profile of selected genes in hiPSCs source cells.

<p>DNA methylation occupancy is reported as the percentage of unmethylated (UM), intermediately methylated (IM) and hypermethylated (HM) DNA. The profile is shown for NDFs (A), ADFs (B), and PBMC CD34⁺ cells (C).</p

DNA methylation profile of selected genes in hPSC lines.

<p>DNA methylation occupancy is reported as the percentage of unmethylated (UM), intermediately methylated (IM) and hypermethylated (HM) DNA. The profile is shown for hESC lines (A), hiPSC lines from NDFs (B), hiPSC lines from ADFs (C, D) and hiPSC lines from PBMC CD34⁺ cells (E, F) generated by STEMCCA lentivirus (STE), Sendai virus (SEN) and the episomal vector (EPI) reprogramming. Values are the averages from hPSC lines within the line group where appropriate.</p

Gene expression levels in hPSCs and dermal fibroblasts, as determined by quantitative real-time PCR.

<p>Expression levels of eleven genes were compared i) among the hiPSC lines, hESC lines and dermal fibroblasts (NDFs and ADFs together) (A-C) and ii) within hiPSC lines between low and high passage numbers (D-F). Analysed hiPSC lines and the passage numbers are reported in the graph legend. The values are relative to the values from hESCs (set at 1). Values are the mean + SD, and asterisks indicate the significance between indicated samples: *p < 0.05; **p < 0.005; ***p < 0.0005.</p

hPSC lines used in this study.

<p>hPSC lines used in this study.</p

Hierarchical clustering of hPSC lines and dermal fibroblasts based on quantitative real-time PCR data.

<p>The colour-coded scale on the left of the picture indicates the expression value for each gene; low expression is represented by green, and high expression is represented by red. The values are relative to the values from CCTL-14 (set at 1).</p

PCR primers used for MethylScreen reactions.

<p>PCR primers used for MethylScreen reactions.</p