116 Chapter 5 Cervical samples in 20 µl Thinprep n = 120 Direct cell conversion protocol Reference protocol Samples are certrifuged for 10 minutes at 4000 rpm 2.5 µl is used in the QIAsure Methylation Test Aliquot of 1000 µl Aliquot of 500 µl DNA concentration measurement with Qubit Pellet is resuspended in 100 µl T1 buffer* and transferred to a deep well plate 480 µl Thinprep is removed and pellet is resuspended in the remaining 20 µl Thinprep 250 ng DNA used for conversion with EZ DNA Methylation Zymo kit Elution in 12.5 µl 20 µl is used for direct conversion with Epitect Fast 96 Bisulphite conversion kit Elution in 50 µl DNA extraction with NucleoMag 96 tissue kit** Elution in 100 µl Invalid results (Cq values ActB >26.4) repeated with 2000 µl Thinprep as input Figure 5.1 Flowchart of the protocols used. * T1 buffer from NucleoMag 96 tissue kit, Macherery-Nagel GmbH&Co. KG, Düren, Germany ** NucleoMag 96 tissue kit, Machery-Nagel GmbH&Co. KG, Düren, Germany Abbreviations: n, number of; Cq, quantification cycle
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