216 Chapter 8 urine. For example, designing assays with an amplicon length below 120 base pairs is crucial for the amplification of short urinary cfDNA fragments. Other considerations for optimal quantitative methylation-specific PCR assay design for liquid biopsies are outlined in Table 1 and stem from both empirical knowledge gained during the course of this thesis and relevant literature (65, 66). Table 1: Considerations for designing optimal multiplex quantitative methylation-specific PCR assays for liquid biopsies. Location selection • Select a clinically relevant differentially methylated region, based on literature, whole-genome sequencing data, or publicly available datasets (e.g. via mexpress.be (67)). • Select a region with a suitable density of CpG sites. Avoid using regions with three or more consecutive CGs. Primer and probe design • Aim for an amplicon size between 60-120 base pairs. • Design primers and probes with at least two methylated Cs each. • Design primers with a potentially methylated C at the 3’-end. • Design primer with a Tm between 58-63 °C. • Design forward and reverse primer with ≤ 2 °C difference. • Design probe with a Tm of 10°C higher than primer Tm. • Avoid probe designs with a G at the 5’-end. • Aim for a GC content between 30-80%. • Check target specificity of primers using BLAST. • Test amplification of the desired amplicon in silico. • Check formation of hairpins, self-dimers, and cross-dimers in silico. • Use LNAs and/or MGB probes to increase the Tm of short primers and probes. Multiplex design • Design primers and probes of different targets with a similar Tm. • Check cross-hybridization of primers and probes of different targets in silico. • Select non-overlapping fluorescent labels for different targets. Primer and probe testing • Validate amplification of the correct product by gel electrophoresis. • Determine optimal primer Tm using gradient PCR reactions. • Ensure PCR efficiencies are close to 100% (range 80-110%) and R2 >0.98. • Refine primer and probe concentrations using primer and probe limiting assays. • Determine optimal cutoff and baseline values per target. Partially adapted from Massen (65) and Snellenberg (66). LNA = locked nuclear acid; MGB = minor groove binding; Tm = melting temperature. 8.3.3 Maximizing the urine cell-free DNA yield The low cfDNA recovery rate from urine poses challenges for downstream molecular analyses. As compared to blood, urine can be collected in large volumes, which could potentially compensate for the low cfDNA yield. However, most existing isolation methods are unable to process large urine volumes, with a maximum urine processing volume
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