An improved quality control for bisulfite-PCR-based DNA methylation analysis: cycle threshold value

被引：4

作者：

Ang, Pei Woon ^{[2
]}

Toh, Hwee Boon ^{[1
]}

Iacopetta, Barry ^{[2
]}

Soong, Richie ^{[1
,3
]}

机构：

[1] Natl Univ Singapore, Oncol Res Inst, Singapore 117456, Singapore

[2] Univ Western Australia, Sch Surg, Nedlands, WA 6009, Australia

[3] Natl Univ Singapore, Dept Pathol, Singapore 117456, Singapore

来源：

CLINICAL CHEMISTRY AND LABORATORY MEDICINE | 2008年 / 46卷 / 08期

关键词：

bisulfite treatment; DNA methylation; epigenetics; molecular diagnostics; quality control; real-time PCR;

D O I：

10.1515/CCLM.2008.217

中图分类号：

R446 [实验室诊断]; R-33 [实验医学、医学实验];

学科分类号：

1001 ;

摘要：

Background: The diagnostic potential of DNA methylation has generated a need for quality control systems in its analysis. Here, we tested whether cycle threshold (Ct) values from real-time quantitative PCR correlate better with the reliability of bisulfite-PCR methylation analyses than spectrophotometric concentrations. Methods: A total of 18 cell line DNA samples were quantified for beta-actin (ACTB) Ct and spectrophotometric values from 1-mu L volumes. A further 1 mu L was analyzed in quintuplicate for bisulfite DNA ACTB by MethyLight. Correlations between Ct and spectrophotometric values, and MethyLight qualitative (detection in all replicates) and quantitative (Ct value standard deviation < 1) reliability were compared. To validate the findings, the same comparisons were made in 40 formalin-fixed, paraffin-embedded tissue (FFPET) samples analyzed by methylation-specific PCR of MLH1. Results: Using optimal thresholds, Ct values correctly identified 6/7 (86%) and 11/11 (100%) cell line samples to be qualitatively, and 7/7 (100%) and 4/4 (100%) quantitatively reliable and unreliable, respectively. The corresponding frequencies for spectrophotometry were 6/8 (75%), 10/10 (100%), 6/8 (75%) and 2/3 (67%), respectively. In FFPET DNA, the respective values for qualitative reliability for Ct values were 25/25 (100%) and 11/15 (73%), and 11/14 (79%) and 8/26 (31%) for spectrophotometry. Conclusions: Ct values could be useful indicators for gauging the suitability of samples for methylation analysis, in particular from FFPET.

引用

页码：1117 / 1121

页数：5

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