Figure 1.
Detection of circulating beta cell DNA in NOD mice using qMSP assay.
A) Blood glucose levels in NOD mice measured every two weeks (n = 5) showing a significant rise in mean blood glucose levels after 16 weeks. The dashed line (200 mg/dL) indicates the hyperglycemic threshold. B) Pancreatic sections of the indicated groups (n = 5) were stained with H&E and the degree of insulitis was scored: no insulitis (white), peri-insulitis (dotted), mild insulitis (hatched), and invasive insulitis (black). C) In parallel, circulating beta cell DNA was measured by qMSP in each mouse group (n = 5) at designated time points. Fold changes in unmethylation are quantified by calculation of the Relative Unmethylation Ration (RUR) for each sample (see Experimental Design and Methods). The data display the mean ± standard error mean (SEM) of three independent measurements. The statistical significance was calculated by unpaired t tests compared with week 8 values and indicated by asterisks (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).
Figure 2.
Tissue-specific methylation of the human INS promoter.
Genomic DNA samples obtained from human blood, breast, colon, kidney, liver, lung, spleen, stomach, and human beta cells (‘Islet cell fraction’≈70% beta cells) were analyzed for methylation of the INS promoter. The positions of the nine CpG sites relative to the transcription starting site (TSS) are indicated. The bars display the position and the percentage of unmethylation (white bars) to methylation (black bars) for each CpG. Each pattern results from 20 to 61 clones and obtained from 6 individuals for blood and 3 individuals for other tissues. Statistics were done using the QUMA computer program and Fisher exact test comparing each site with the same site in beta cells. The statistical significance is indicated by asterisks (*, p<0.1; **, p<0.01).
Figure 3.
Tissue methylation pattern of the human INS exon 2.
The human tissues shown in Figure 2 were analyzed for methylation of INS exon 2 (8 CpG), intron 1 (2 CpG) and intron 2 (2 CpG). The positions of the twelve CpG sites relative to the transcription starting site (TSS) are indicated. The bars display the position and percentage of unmethylation (white bars) to methylation (black bars) for each CpG. Each pattern results from 5 to 35 clones obtained from 5 individuals blood, 2 individuals beta cells and breast, and 3 individuals for the other tissues. Statistics were done using the QUMA computer program and Fisher exact test comparing each site with the same site in beta cells. The statistical significance is indicated by asterisks (*, p<0.1; **, p<0.01).
Figure 4.
Primer selection and analytical performance of methylation-specific PCR.
A) Schematic illustration of the human INS gene promoter region showing the position of the nine CpG sites. Solid arrows represent the bisulfite-specific primers (BSPs) that amplify both methylated and unmethylated DNA. Dashed arrows represent methylation-specific primers (MSPs) that amplify unmethylated DNA only. B) Unmethylated plasmid was serially diluted after bisulfite conversion and analyzed by qMSP using selected primer sets. Agarose gel electrophoresis of MSP reactions showing the size of the PCR products. C) Graphs of real-time SYBR Green PCR data showing linearity of Cq versus log copy number of unmethylated plasmid (averages and standard deviation (SD)) from 5 to 106 copies.
Table 1.
Statistical variation of qMSP standard curve.
Table 2.
The amplification efficiency of qMSP and qBSP standard curves.
Figure 5.
Serial dilutions of the bisulfite-converted gDNA obtained from human islets, blood, spleen and colon were used as a template for nested PCR using either BSP (A) or MSP (B) in the first-step reaction. The products were used as a template for the second-step MSP reaction. The data display the mean ± SEM of the Relative Unmethylation Ratio (RUR). The cloned INS promoter was used for normalization and standardization of the results. Statistically significant differences at each DNA concentration between islets and other tissues were calculated using two way ANOVA and the significance level indicated by asterisks (****, p<0.0001; ***, p<0.001; **, p<0.01).
Figure 6.
Quantitative MSP for monitoring beta cells in islet transplant patients.
(A) Blood samples were collected from 6 normal healthy individuals and pre- and post- transplantation (at days 1 and 14 post-TX) from 6 islet transplant patients. Genomic DNA from the samples was bisulfite-converted and used for nested qMSP and BSP assays. The data display the mean ± SEM of the Relative Unmethylation Ratio (RUR) calculations. (B) Plasma samples were prepared from the islet recipient blood samples and analyzed as in A. The statistical significance was calculated with the Wilcoxon test to compare RUR of samples after transplant with that before transplant and significance level indicated by asterisks (*, p<0.05; **, p<0.01).