Molecular testing was performed blinded for cytology and histology outcomes at the Department of Pathology of Amsterdam UMC. The QIAscreen HPV PCR Test (Qiagen) was used for high-risk HPV testing with separate genotyping for HPV16, HPV18, and a pool of 13 other high-risk HPV types (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67, and 68). The QIAsure Methylation Test (Qiagen) was used for FAM19A4/miR124-2 methylation testing on bisulfite-converted DNA. The methylation test result was labeled positive if the QIAsure Methylation Test result exceeded the preset threshold for methylation positivity. Baseline clinician-collected samples stored in SurePath (n = 11) were not tested for FAM19A4/miR124-2 methylation because of insufficient DNA quality after DNA isolation.
Qiasure methylation test
Manufactured by Qiagen
Sourced in Germany
The QIAsure Methylation Test is a laboratory equipment product designed for the analysis of DNA methylation patterns. It provides a standardized approach to quantify and evaluate the methylation status of specific genomic regions. The core function of this product is to enable the assessment of DNA methylation levels, which can serve as a molecular biomarker for various applications.
Automatically generated - may contain errors
5 protocols using qiasure methylation test
1
HPV and Methylation Testing Protocol
2
Quantitative Assessment of FAM19A4/miR124-2 Methylation
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FAM19A4/miR124‐2 methylation analysis including sample DNA extraction was performed as previously described.24 For bisulfite‐conversion, the EZ DNA Methylation Kit was used according to the manufacturer's specifications (Zymo Research, Irvine, California).32 Bisulfite‐converted DNA was subsequently used as input for quantitative PCR analysis of the FAM19A4 and miR124‐2 promoter methylation levels using the QIAsure Methylation Test (QIAGEN). For all centers, a sample input of 2.5 μL bisulfite‐converted DNA was used for PCR on the Rotor‐Gene Q MDx 5plex HRM instrument (QIAGEN) equipped with the AssayManager software (QIAGEN). This software runs the assay followed by automatic quality assurance and data analysis. The reported results were hypermethylation‐positive, negative or invalid. Additional quality assurance was employed using the housekeeping gene β‐actin (ACTB) as a reference for successful bisulfite‐conversion, sample quality and signal normalization. Methylation testing was performed by local technicians blinded for the clinical data.
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FAM19A4/miR124‐2 methylation analysis including sample DNA extraction was performed as previously described.
3
HPV and Methylation Analysis for Cervical Disease
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HPV status and FAM19A4/miR124‐2 methylation analysis were evaluated in cervical scrapes corresponding to the selected tissue blocks. CIN lesions were only included in case the corresponding cervical scrape was high‐risk HPV positive. Participating institutes used clinically validated high‐risk HPV DNA assays to determine HPV status26 and used the QIAsure Methylation Test (QIAGEN, Hilden, Germany) on the Rotorgene PCR‐platform (QIAGEN, Hilden, Germany) to assess FAM19A4/miR124‐2 methylation status as described previously.25 , 27 The housekeeping gene β‐actin (ACTB) was used as a reference gene. Methylation levels were calculated as ΔΔCt values by comparing the target Ct values to the Ct values of ACTB relative to that of the calibrator. Methylation status was labelled positive if the QIAsure Methylation Test result exceeded the preset ΔΔCt value threshold for methylation positivity for FAM19A4 and/or miR124‐2 according to manufacturer's instructions.
4
QIAsure Methylation Test for CIN3+ Detection
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Bisulfite‐converted DNA was used as input for QIAsure Methylation test (Qiagen). The standard DNA input was 2000 ng, but for samples with reduced DNA concentrations, as low as 100 ng was used. The methylation test was performed according to the manufacturer's instructions, where 2.5 μL bisulfite‐converted DNA was used as input for the PCR, conducted on the Rotor‐Gene Q MDx 5plex HRM instrument (Qiagen). The ASSAYMANAGER software enables automated interpretation of the results and uses the housekeeping gene β‐actin (ACTB) as a quality control and reference for successful bisulfite‐conversion. The software calculates the ddCt values for both FAM19A4 and hsa‐miR124‐2, and a sample is positive for hypermethylation if either FAM19A4 or hsa‐miR124‐2 or both are above the cutoff point. When ACTB was not detected, the analysis was invalid according to the manufacturer's instructions.
LBC samples (n = 113) from women who developed CIN3+ were analyzed, along with 31 healthy controls (described above).
LBC samples (n = 113) from women who developed CIN3+ were analyzed, along with 31 healthy controls (described above).
5
Cervical Methylation Biomarker Analysis
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FAM19A4/miR124-2 methylation analysis was performed previously, blinded for cytology and histology outcomes, by quantitative methylation specific PCR (qMSP) on bisulphite converted DNA from cervical scrapes collected at baseline using a prototype version of the QIAsure Methylation Test® (Qiagen, Hilden, Germany) [20] (link).
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