Computational protocol: Applicability of HIN-1, MGMT and RASSF1A promoter methylation as biomarkers for detecting field cancerization in breast cancer

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Protocol publication

[…] DNA extracted from biopsy samples and human control DNA (fully methylated and unmethylated) were treated with sodium bisulfite using the EpiTect Fast DNA Bisulfite Kit (Qiagen) according to the manufacturer’s instructions. Fully methylated control DNA (CpGenom Universal Methylated DNA) was obtained from EMD Millipore (Billerica, MA, USA), unmethylated control DNA (EpiTect Control DNA (human), unmethylated) from Qiagen.Primers for CCND2 (GenBank: CM000263.1), MGMT (GenBank: X61657.1) and RASSF1A (GenBank: NG_023270.1), targeting CpG island regions flanking the transcription site, were designed with the Methyl Primer Express Software v1.0 (Applied Biosystems, Carlsbad, CA, USA). Primer sequences for DAPK1 [], GSTP1 [] and HIN-1 [] were taken from the literature. For each MS-HRM method, the annealing temperature (Ta) and the MgCl2 concentration were optimized in-house. Primer sequences and optimized conditions are summarized in Table .Polymerase chain reaction (PCR) amplification of the bisulfite-treated DNA and HRM analysis were performed using a Rotor-Gene Q thermocycler (Qiagen). Each reaction mixture had a total volume of 20 μl, containing 10 ng of bisulfite-treated DNA, 10 μl of 2× EpiTect HRM PCR Master Mix (Qiagen), forward and reverse primer and RNase-free water. In all PCR reactions, the concentration of forward and reverse primer was 250 nM each. PCR amplification was carried out under the following conditions: initial step at 95 °C for 5 min; followed by 50 cycles at 95 °C for 10 s, Ta of the respective primer set for 30 s and 72 °C for 10 s (touchdown 1 °C, 7 cycles); denaturation step at 95 °C for 1 min followed by a hybridization step at 40 °C for 1 min. In the HRM step, the temperature was increased by 0.1 °C increments per 2 s.HRM data were evaluated with the Rotor-Gene Q Series Software 2.1.0 (Qiagen). Each biopsy sample was analyzed at least twice in duplicate. The DNA methylation status was calculated with the help of calibration curves established by analyzing mixtures of fully methylated and unmethylated human control DNA. In order to obtain accurate results also for heterogeneously methylated sequences, an interpolation calibration curve was established as proposed by Migheli et al. []. However, we slightly changed their approach and calculated the average of the normalized fluorescence signal for each standard over the entire temperature interval instead of using single values at chosen temperature points. Calibration functions were established with SigmaPlot 11.0 (Systat Software Inc., San Jose, CA, USA). Limit of detection (LOD) and limit of quantification (LOQ) of the MS-HRM methods were determined by repeatedly analyzing bisulfite-treated, unmethylated control DNA. After calculating the mean and the standard deviation, the LOD (signal-to-noise ratio (S/N) of 3) was determined by adding three times the standard deviation and the LOQ (S/N of 10) by adding ten times the standard deviation to the mean. The corresponding methylation status was then calculated with the help of the equation of the calibration curves established by analyzing mixtures of methylated and unmethylated control DNA. [...] Statistical analyses were carried out with IBM SPSS Statistics 21.0 (IBM Corp., Armonk, NY, USA). The methylation status was treated either as categorical variable (< LOD, < LOQ or ≥ LOQ) or as continuous variable. If the methylation status was treated as continuous variable, methylation status < LOD and < LOQ were substituted with a default value, namely half the LOD and half the LOQ, respectively, as proposed previously []. Chi-squared test was used to evaluate if the methylation status of the six genes is associated with any of the clinicopathological parameters. Independent-samples t test was applied to evaluate if there are significant differences in the methylation status between the tumor tissues and noncancerous breast tissues of the control group. One-way ANOVA (analysis of variance), followed by Tukey’s test, was applied to test for significant differences in the DNA methylation status between tumor, tumor-adjacent and tumor-distant tissues. Levene’s test was used to assess the equality of variances. Pearson’s correlation coefficient was used to assess the relationship between two continuous variables. In all cases, p < 0.05 (two-sided) was considered significant. […]

Pipeline specifications

Software tools SigmaPlot, SPSS
Application Miscellaneous
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms, Cardiovascular Abnormalities