Direct bisulphite conversion of cervical samples for DNA methylation analysis 117 5 RESULTS To assess whether bisulphite conversion directly on cervical cells performs equally well as the reference protocol, 120 cervical samples were subjected to both protocols. For the reference protocol, genomic DNA was isolated and DNA concentrations measured. DNA yield of the samples varied between 0.18 μg and 9.75 μg. Accordingly, 108/120 (90.0%) samples reached the normalised input of 250 ng for bisulphite conversion. All 120 samples had a valid test result, with ACTB Cq values ranging from 22.53-26.11. With 1/40th sample input volume in the direct conversion protocol, 114/120 (95.0%) had a valid test result, with ACTB Cq values ranging from 20.67-26.26. Of the six initially invalid samples (ACTB Cq values 26.41-31.77), five samples had sufficient material available for repeat testing with 1/10th sample input volume, which all generated valid test results, with ACTB Cq values ranging from 24.48-25.68. ΔΔCq values of FAM19A4 and miR124-2 were significantly correlated between both protocols (Figure 5.2A and 5.2B; Spearman Rho 0.708 p value = 0.000 and 0.763, p value = 0.000, respectively), with particularly strong correlation in CIN2+ (Spearman’s Rho 0.965, p value = 0.000 for FAM19A4, and 0.889, p value = 0.000 for miR124-2). In further investigation of the agreement between methylation results from each protocol, Bland-Altman plots were prepared (Figure 5.2C and 5.2D). Overall there was a good agreement between ΔΔCq values obtained using the direct cell conversion protocol and the reference protocol for both FAM19A4 (Figure 5.2C) and miR124-2 (Figure 5.2D), with a few outliers observed mainly at higher pairwise average ΔΔCq values. DISCUSSION In this study, we report on a direct cell bisulphite conversion protocol that can be performed on cervical samples, without the need of prior genomic DNA isolation and input normalisation. The direct cell conversion protocol had a high valid rate and performed analytically equally well on cervical samples as the reference protocol using normalised DNA as input. Bisulphite conversion is an essential step in many DNA methylation assays. A direct cell conversion protocol may overcome some laborious steps in the workflow of DNA methylation analysis, i.e., genomic DNA isolation, DNA concentration measurement and standardisation of DNA input, and thereby is of great interest for implementation
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