In:
Medical Epigenetics, S. Karger AG, Vol. 2, No. 1 ( 2014-2-14), p. 28-36
Abstract:
〈 b 〉 〈 i 〉 Introduction: 〈 /i 〉 〈 /b 〉 Bisulfite treatment of DNA introduces methylation-dependent sequence changes through selective chemical conversion of nonmethylated cytosine to uracil and serves as pretreatment step for the majority of DNA methylation analysis methods. 〈 b 〉 〈 i 〉 Methods: 〈 /i 〉 〈 /b 〉 We have evaluated the conversion performance of five of the most commonly used bisulfite treatment kits [MethylDetector (Active Motif), Epitect+ (Qiagen), Zymo Methylation, Zymo Gold and Zymo Lightning (all from Zymo Research)] by pyrosequencing four different regions with variable methylation levels, including: a repetitive element 〈 i 〉 (ALUSX) 〈 /i 〉 , a gene with low levels of methylation 〈 i 〉 (IL6ST) 〈 /i 〉 , an imprinted gene expected to be approximately 50% methylated 〈 i 〉 (IGF2) 〈 /i 〉 , and a fully methylated gene 〈 i 〉 (ST3GAL2) 〈 /i 〉 . In addition, we have studied the influence of duration (3 vs. 16 h) and type (fixed temperature vs. cycling program) of incubation protocol on the conversion efficiency of each evaluated kit. 〈 b 〉 〈 i 〉 Results: 〈 /i 〉 〈 /b 〉 All kits produced similar conversion rates of 〈 i 〉 ALUSX, 〈 /i 〉 〈 i 〉 IGF2 〈 /i 〉 and 〈 i 〉 ST3GAL2 〈 /i 〉 , while the conversion of the low methylated 〈 i 〉 IL6ST 〈 /i 〉 gene was variable between kits. The Zymo kits were highly consistent in their performance even when different protocols of incubation were applied, generating full conversion at the low methylated gene 〈 i 〉 IL6 〈 /i 〉 ; this was not true for the MethylDetector and Epitect+ kits. However, long-cycling incubation could produce similar conversion rates for the same locus in combination with Active Motif and Qiagen kits. 〈 b 〉 〈 i 〉 Conclusions: 〈 /i 〉 〈 /b 〉 The selection of a long-cycling protocol during conversion permits standardization of protocols, improving the reproducibility of methylation estimates across laboratories for gene-specific, genome-wide and bisulfite-based sequencing analyses.
Type of Medium:
Online Resource
ISSN:
1664-5561
Language:
English
Publisher:
S. Karger AG
Publication Date:
2014
detail.hit.zdb_id:
2704578-X
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