Direct bisulphite conversion of cervical samples for DNA methylation analysis

ABSTRACT

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.

KEYWORDS:

• Sodium bisulphite treatment
• high-throughput
• host-cell DNA methylation
• HPV triage
• cervical screening
• liquid-based cytology

Acknowledgments

We would like to thank Ms Elia Alcaniz Boada (Scottish HPV Archive) for preparation of samples for the study.

Author contributions

Study design: DAMH, RDMS, AF, AH, CJLMM

Data collection: SD, RB, KC

Data management: LV, AF

Laboratory experiments: SD

Statistical analysis: LV

Data interpretation: LV, DAMH

Writing first draft of manuscript: LV, DAMH

All authors were involved in writing the manuscript and gave final approval of the submitted and published version of the manuscript.

Data availability

The data that support the findings of our study are available from the corresponding author upon reasonable request.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was funded by the a grant from the Dutch Cancer Society (KWF Kankerbestrijding 11337) and SME Instrument in the Horizon 2020 Work Program of the European Commission (Valid-screen 666800).