TY - JOUR
T1 - Direct bisulphite conversion of cervical samples for DNA methylation analysis
AU - Verhoef, Lisanne
AU - Floore, Arno
AU - Doorn, Saskia
AU - Cuschieri, Kate
AU - Bhatia, Ramya
AU - Hesselink, Bart
AU - Meijer, Chris J L M
AU - Steenbergen, Renske D M
AU - Heideman, Daniëlle A M
N1 - Publisher Copyright:
© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
PY - 2021/10/15
Y1 - 2021/10/15
N2 - Sodium bisulphite conversion of DNA to separate methylated from unmethylated cytosines is a standard for methylation analysis. This study evaluated a direct cell conversion protocol on cervical samples as alternative to isolated genomic DNA as input. Clinician-collected cervical samples (n = 120) were subjected to a direct conversion protocol, or genomic DNA was isolated with a fixed amount used for subsequent bisulphite conversion. Converted samples were compared for ACTB control gene and methylation of FAM19A4 and miR124-2 genes using quantitative methylation-specific PCR (QIAsure Methylation Test). Direct conversion resulted in a high success rate, i.e., 119/120 (99.2%) samples reported a valid test result. ΔΔCq values of FAM19A4 and miR124-2 were significantly correlated between both protocols (Spearman Rho 0.708 and 0.763, respectively, all p-values = 0.000). Agreement between both the bisulphite protocols was demonstrated by Bland–Altman plots. A direct cell conversion protocol shows good technical and analytical performance and offers a streamlined workflow for methylation analysis.
AB - Sodium bisulphite conversion of DNA to separate methylated from unmethylated cytosines is a standard for methylation analysis. This study evaluated a direct cell conversion protocol on cervical samples as alternative to isolated genomic DNA as input. Clinician-collected cervical samples (n = 120) were subjected to a direct conversion protocol, or genomic DNA was isolated with a fixed amount used for subsequent bisulphite conversion. Converted samples were compared for ACTB control gene and methylation of FAM19A4 and miR124-2 genes using quantitative methylation-specific PCR (QIAsure Methylation Test). Direct conversion resulted in a high success rate, i.e., 119/120 (99.2%) samples reported a valid test result. ΔΔCq values of FAM19A4 and miR124-2 were significantly correlated between both protocols (Spearman Rho 0.708 and 0.763, respectively, all p-values = 0.000). Agreement between both the bisulphite protocols was demonstrated by Bland–Altman plots. A direct cell conversion protocol shows good technical and analytical performance and offers a streamlined workflow for methylation analysis.
KW - HPV triage
KW - Sodium bisulphite treatment
KW - cervical screening
KW - high-throughput
KW - host-cell DNA methylation
KW - liquid-based cytology
UR - http://www.scopus.com/inward/record.url?scp=85118356695&partnerID=8YFLogxK
U2 - 10.1080/15592294.2021.1992911
DO - 10.1080/15592294.2021.1992911
M3 - Article
C2 - 34652264
JO - Epigenetics
JF - Epigenetics
SN - 1559-2294
ER -