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J Mol Diagn. 2010 Mar;12(2):177-83. doi: 10.2353/jmoldx.2010.090106. Epub 2010 Jan 21.

Precision of pyrosequencing assay to measure LINE-1 methylation in colon cancer, normal colonic mucosa, and peripheral blood cells.

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  • 1Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, 44 Binney St., Room JF-215C, Boston, MA 02115, USA.

Abstract

Genome-wide DNA hypomethylation plays an important role in epigenomic and genomic instability and colorectal carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. In addition, LINE-1 hypomethylation in blood cells has been associated with colorectal adenoma risk, and LINE-1 hypomethylation in colorectal cancer is related with prognosis and linearly predicts shorter patient survival. However, no study has comprehensively evaluated the precision of sodium bisulfite conversion and PCR-pyrosequencing to measure LINE-1 methylation. Using 10 paraffin-embedded colon cancers, 5 matched normal colon mucosa, and 5 unrelated peripheral blood buffy coat leukocyte specimens, we enriched tumor DNA by macrodissection and laser capture microdissection. LINE-1 methylation was calculated as an average of 100 * C/(C + T) at 4 CpG sites after bisulfite-PCR-pyrosequencing. The LINE-1 methylation value in colon cancers varied, ranging approximately from 30 to 80. To measure assay precision, we performed bisulfite conversion on seven different DNA specimen aliquots and repeated PCR-pyrosequencing seven times. Run-to-run (between-run) SD ranged from 1.3 to 4.4 (median, 3.0) in macrodissected colon cancers; 1.1 to 10.5 (median, 3.8) in laser capture microdissection specimens; 1.3 to 2.5 (median, 1.9) in normal colon; and 1.5 to 3.4 (median, 1.9) in leukocyte DNA. In conclusion, bisulfite conversion and PCR-pyrosequencing assay can measure LINE-1 methylation in macrodissected colon cancer, normal colon, and blood DNA, and may be useful in clinical and research settings.

PMID:
20093385
[PubMed - indexed for MEDLINE]
PMCID:
PMC2871724
Free PMC Article
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