Precision of Pyrosequencing Assay to Measure LINE-1 Methylation in Colon Cancer, Normal Colonic Mucosa, and Peripheral Blood Cells Natsumi Irahara, Katsuhiko.

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Precision of Pyrosequencing Assay to Measure LINE-1 Methylation in Colon Cancer, Normal Colonic Mucosa, and Peripheral Blood Cells Natsumi Irahara, Katsuhiko Nosho, Yoshifumi Baba, Kaori Shima, Neal I. Lindeman, Aditi Hazra, Eva S. Schernhammer, David J. Hunter, Charles S. Fuchs, Shuji Ogino The Journal of Molecular Diagnostics Volume 12, Issue 2, Pages 177-183 (March 2010) DOI: 10.2353/jmoldx.2010.090106 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Pyrosequencing to measure LINE-1 methylation. A: Microdissected colon cancer (Case #1). B: Microdissected colon cancer (Case #2). The “%” numbers (in dark shade) are proportions of C and T at each CpG site after bisulfite conversion. Thus, the methylation level of each CpG site is estimated by the proportion of C (%). An overall LINE-1 methylation level is calculated as the average of the proportions of C at the four CpG sites. The first, third and fourth CpG sites follow stretches of Ts, resulting in higher T peaks (in light shade) than the second CpG site, and the proportion of C has been adjusted accordingly. The arrows indicate no residual C at the non-CpG site, ensuring complete bisulfite conversion. The Journal of Molecular Diagnostics 2010 12, 177-183DOI: (10.2353/jmoldx.2010.090106) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Strategy to assess precision of bisulfite conversion and PCR-pyrosequencing on each specimen (an example of macrodissected colon cancer Case #1). Bisulfite conversion was performed on seven different aliquots (B1 through B7) from each specimen. PCR-pyrosequencing was performed on the 7 bisulfite-treated specimens (B1–B7), and was repeated seven times on two specimens (B1 and B2) on seven different days (P1 through P7). The Journal of Molecular Diagnostics 2010 12, 177-183DOI: (10.2353/jmoldx.2010.090106) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Results on repeated measurements of LINE-1 methylation levels. A: Bisulfite-to-bisulfite variation of LINE-1 methylation values. Each dot and vertical bar represent mean and SD [SD or SD(B) in Table 1], respectively, of LINE-1 methylation values among different bisulfite-treated DNA specimens from each case (DNA source). The x-axis indicates DNA sources, ie, 10 macrodissected colon cancers, 5 matched colon cancers collected by LCM, 5 matched normal colons, and 5 unrelated blood DNA specimens, with each number representing Case ID. B: Run-to-run (between-run) variation of LINE-1 methylation values. Each dot and vertical bar represent mean and SD [SD or SD(P) in Table 1], respectively, of LINE-1 methylation values among different runs on the same bisulfite-treated DNA specimen. The x-axis indicates DNA sources, ie, 10 macrodissected colon cancers, 5 matched colon cancers collected by LCM, 5 matched normal colons, and 5 unrelated blood DNA specimens, with each number representing Case ID. Results from B1 (bisulfite-treated specimen #1; see Figure 2) and B2 are shown side-by-side for each DNA source. C: Mean LINE-1 methylation value and SE of mean in the 5 macrodissected colon cancers and matched LCM specimens. Note that each SE of mean is small due to seven repeated measurements. When a LCM specimen showed low-level LINE-1 methylation (eg, Cases 6 and 7), a matched macrodissected specimen also showed low-level LINE-1 methylation. The Journal of Molecular Diagnostics 2010 12, 177-183DOI: (10.2353/jmoldx.2010.090106) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions