Direct bisulphite conversion of cervical samples for DNA methylation analysis 115 5 DNA METHYLATION ANALYSIS FAM19A4/miR124-2 methylation analysis was performed using the QIAsure Methylation Test (QIAgen, Hilden, Germany) according to the manufacturer’s instructions 14. Sample input in the assay, for both the reference and direct cell conversion protocol, is 2.5 μl bisulphite-converted DNA. The assay was performed on a Rotor-Gene Q Mdx 5plex HRM instrument (QIAgen, Hilden, Germany). AssayManager software (QIAgen, Hilden, Germany) controls amplification as well as data analysis and reporting using a fixed assay profile. The housekeeping gene β-Actin (ACTB) was used to verify DNA quality and successful bisulphite conversion. A sample was considered to have a valid test result when Cq value of ACTB was below 26.4. ΔCq values were calculated for each target separately (i.e., FAM19A4 and miR124-2) as the difference between the Cq value of the target and the Cq value of the reference (ACTB). This ΔCq is a relative quantitative value for the promotor methylation level of the FAM19A4 or miR124-2 gene. For normalisation, the ΔCq value of a calibrator sample that is included in the QIAsure Methylation Test, was subtracted from the ΔCq of the target resulting in a ΔΔCq value. The calibrator is a standardised low-copy plasmid DNA sample with known copy number of the three targets (i.e., FAM19A4, miR124-2 and ACTB). A lower ΔΔCq value corresponds to a higher methylation level of the respective target. DATA AND STATISTICAL ANALYSIS The direct cell bisulphite conversion protocol was compared with the reference protocol for results of ACTB control gene (Cq values) and methylation of FAM19A4 and miR124-2 genes (ΔΔCq values). Paired evaluation of ΔΔCq values of FAM19A4 and miR124-2 for both bisulphite protocols was done by Spearman rank analysis. Agreement between ΔΔCq values derived from the two protocols was visualised using Bland-Altman plots. In these plots the difference of two methods is plotted against the average of both methods. We computed the mean of the differences (i.e., bias), the standard deviation (SD) and the 95% limits of agreement (= bias ±1.96 × SD) 16. All statistical analyses were performed in IBM SPSS Statistics version 26.0 (International Business Machines Corp., Armonk, New York, USA) and p values are two-sided with 0.01 as significance threshold.
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