Methylation vs Expression in Promoter regions.

<p>Analysis of DM loci within promoter regions and their overlap with differential gene expression. (Left panel A) All 21 pairs (all NSCLC histologies), overall differences do vary by PR genomic location (CGI, CGS, other), ChiSquare p = 3.32E-4. <i>(Right panel B)</i> Within the set of adenocarcinomas overall differences do vary by PR genomic location (CGI, CGS, other), ChiSquare p = 1.10E-7. Majority of DM promoter loci are associated with hypermethylation when the DM loci are within CG islands. This effect is more pronounced among adenocarcinomas, where the DM loci in CG islands are mostly associated with downregulation of the gene. KEY: “M” = methylation, “E” = expression. Upward arrow indicates increase and downward arrow indicates decrease.</p

Heat Map of the Top 50 DM Loci within Adenocarcinomas.

<p>(A) Promoter regions; and (B) Gene body regions. Several genes show differential methylation (DM) at more than one locus and appear multiple times in the heatmap. Blue = Non Tumor, Red = Tumor.</p

The genome compartment represented on the HELP Nimblegen microarray and statistically significant DM loci.

<p>(A) Approximately 91% of the 1.2 million loci represented on the HELP microarray are located in gene body (GB) and intergenic (IG) regions, with a small minority (9%) of the loci located within promoters (PR). (B) Statistical significance (Y-axis) vs. delta (X-axis) (magnitude) of DM. Delta (X-axis) indicates the difference in methylation between tumor (T) vs non-tumor (NT) at a given locus. Loci hypermethylated in T relative to NT have delta < 0. P-value (Y-axis) is calculated based on Benjamini Hochberg adjusted FDR. At FDR p < 0.05, 433,505 loci across all genomic compartments are found to be differentially methylated in T vs NT. Red dots indicate statistically significant DM loci.</p

Magnitude and Direction of differential methylation and its distribution across genomic compartments.

<p>(A) All NSCLC histologies DM was classified as negligible, small or moderate based on the absolute value. (1< abs delta <2 is Moderate/Large; 1< abs delta <0.5 is Small; 0.5< abs delta <0 is Negligible). DM loci with FDR p<0.05 based on paired T-test were considered for this analysis. Majority of hypermethylation in tumors is observed to be of moderate/large magnitude in promoters and gene bodies, while in the intergenic regions, small changes are most frequent. The majority of hypomethylation is observed to be of small magnitude in all the three compartments. A significant fraction of hypomethylation changes are of negligible magnitude yet statistically significant. (B) Direction of DM and the distribution within promoters categorized based on location within CG-islands and CG-shores. Within the category of DM promoter loci, hypermethylation is more frequent in tumors as compared to hypomethylation for those loci within CG-islands and CG-shores. Overall DM differences do vary by PR genomic location (CGI, CGS, other); all NSCLC histologies were ChiSquare p = 2.2E-16; adenocarcinoma-only histology ChiSquare p = 1.9E-4.</p

Titration of lyophilized human recombinant glucose-6-phosphate dehydrogenase (r-G6PD).

<p>Lyophilized human r-G6PD (blue line) was titrated and tested in the Trinity Biotech quantitative assay alongside the Trinity controls (grey line). From these data, three lyophilized concentrations were chosen to represent normal, intermediate, and deficient enzyme activity.</p

Workflow for the use of the lyophilized control with a glucose-6-phosphate dehydrogenase (G6PD) rapid diagnostic test.

<p>Workflow for the use of the lyophilized control with a glucose-6-phosphate dehydrogenase (G6PD) rapid diagnostic test.</p

Stability of the reconstituted lyophilized recombinant glucose-6-phosphate dehydrogenase (r-G6PD).

<p>Lyophilized controls were reconstituted under four conditions to determine stability after rehydration. Three concentrations were assessed: normal (blue), high intermediate (purple), and intermediate (green). The controls were rehydrated in protein storage buffer (PSB) or deionized (DI) water and kept on ice or at room temperature after rehydration: PSB on ice (filled squares), PSB at room temperature (filled circles), DI water on ice (filled triangles), and DI water at room temperature (filled diamonds). Activity was measured on the Trinity Biotech quantitative assay at time zero, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and overnight 24 hours later. Thresholds are shown to indicate whether each measurement is still within an acceptable range of G6PD enzyme activity based on the ranges of Trinity controls: lower acceptable normal (blue dotted line), upper intermediate (black dotted line), lower intermediate (green dotted line), and upper deficient (red dotted line).</p

Stability of lyophilized human recombinant glucose-6-phosphate dehydrogenase (r-G6PD) reagent panel: Normal (blue), intermediate (green), and deficient (red).

<p>Lyophilized panel of three concentrations of human r-G6PD were stored for 12 months at five different temperatures: -80°C filled squares, 4°C filled circles, 30°C filled diamonds, 45°C filled triangles, and 55°C crosses. Activity was measured using the Trinity Biotech quantitative assay. The graph shows the stability of the panel over time. Stability starts to decline after approximately one month at a high temperature of 55°C. Thresholds are shown to indicate whether each measurement is still within an acceptable range of G6PD enzyme activity based on the ranges of Trinity controls: lower acceptable normal (blue dotted line), upper intermediate (black dotted line), lower intermediate (green dotted line), and upper deficient (red dotted line).</p

Performance of human recombinant glucose-6-phosphate dehydrogenase (r-G6PD) controls on qualitative tests for G6PD.

<p>Two lots of three concentrations (normal, intermediate, and deficient) of human r-G6PD were tested on two qualitative assays for G6PD: the fluorescent spot test (panels A and B) and a novel prototype rapid diagnostic test for G6PD (panels C and D). In panels A and B, the top row represents the Trinity normal controls for reference; the second, third, and fourth rows show the signals for the normal, intermediate, and deficient human r-G6PD controls, respectively. In panels C and D, the line intensity of test output correlates with expected enzyme activity, referring to normal, intermediate, and deficient activity. The signal for one lot of freshly lyophilized human r-G6PD (A and C) is compared to a second lot that had been stored at 4°C for more than one year (B and D).</p

Comparison of freshly lyophilized recombinant glucose-6-phosphate dehydrogenase (r-G6PD) versus year-old lyophilized r-G6PD and control.

<p>Human r-G6PD was lyophilized and assayed as “fresh” (grey bars) and compared to lyophilized r-G6PD samples from a lot that had been stored at 4°C for one year (striped bars). The Trinity controls were run at the same time for comparison of activity levels (black bars).</p