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Supplementary material
Methods
Methylprofiler
For identification of relevant promoter methylation we analyzed the colorectal cancer cell
lines SW480 and Caco-2 using two Methyl-Profiler DNA Methylation PCR Array Systems
(Qiagen, Hilden, Germany) which enables fast and accurate detection of DNA methylation
status at CpG islands. The Human Colon Cancer Methyl-Profiler DNA Methylation PCR Array
profiles the methylation status of 24 tumor suppressor gene promoters whose
hypermethylation has been frequently reported in colon tumors. The Human WNT Signaling
Pathway Methyl-Profiler PCR Array analyzes the promoter methylation status of a panel of
24 promoters of genes involved in WNT signaling during carcinogenesis and cellular
differentiation. The assays were performed according to the manufacturer’s instructions. In
brief, following digestion of DNA from SW480 and Caco-2 with methylation-sensitive and dependent restriction enzyme, the remaining input DNA was quantified with real-time PCR
in each individual enzyme reaction using primer that flank the promoter region of interest.
Data analysis is based on the ΔΔCt method with normalization of the raw data to either
housekeeping genes or an external RNA control and was performed with the EpiTect Methyl
II PCR Data Analysis software available from Qiagen (Hilden, Germany). EpiTect Methyl II
PCR Arrays provide gene methylation status as percentage unmethylated (UM) and
percentage methylated (M) fraction of input DNA. "UM" represents the fraction of input
genomic DNA containing no methylated CpG sites in the amplified region of a gene. "M"
represents the fraction of input genomic DNA containing two or more methylated CpG sites
in the targeted region of a gene. The number of CpG sites methylated in a targeted region
can vary within the fraction of methylated input DNA.
DNA extraction from FFPE and serum
DNA was extracted from the paraffin-embedded tumor samples as follows. Briefly, formalinfixed, paraffin-embedded tissue samples were sliced into 5-µm-thick sections and manually
dissected by a pathologist to reduce the proportion of non-tumor cells in the samples and
scraped into an Eppendorf tube. The minimum percentage of tumor cells was 40%. Slides
were deparaffinized twice with 1ml xylene and centrifuged at 13.000 rpm at RT for 10
minutes. Samples were washed with graded ethanol (100%, 90%, 70%) and air dried at 37°C
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Heitzer et al
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for 10 minutes. 200µl of Lysis Buffer (4M Urea, 200mM Tris, 20mM NaCl, 200mM EDTA; pH
7.4) was added and samples were incubated at 98°C for 5 minutes. After spinning down the
samples another incubation step at 98°C for 25 minutes followed. 40µl of Proteinase K
(20mg/ml; Qiagen, Hilden Germany) were added and samples were incubated over night at
57°C. On the next day samples were incubated with another 20µl of Proteinase K until the
samples were completely lysed. Proteinase K was inactivated at 98°C for 10 minutes. The
lysate was transferred to a new tube without carrying over any cell debris. DNA was
precipitated with 200µl Isopropanol at -20°C over night. The DNA pellet was washed with
500µl 70% Ethanol, air dried and dissolved in 20µl TE buffer.
Methylation analysis
DNA derived from tumor samples is mainly of low quality and the concentration is often
overestimated by simple absorbance measurement. In addition, FFPE material itself,
especially when bisulfite converted, represents a poor substrate for PCR amplification. Since
the analysis of the methylation status was carried out through high molecular, fully
methylated DNA, an accurate determination of the amplifiable DNA concentrations was
necessary. For this reason we introduced a normalization step using ALU PCR and assigned
each sample to five different categories, depending on the Ct values of the ALU PCR.
In brief, we used the mean Ct values of different amounts of universally methylated DNA
(100%M) (5ng, 0.5ng, 0.05ng, 0.01ng) of at least 6 independent runs (mean CV of all
categories 0.018, range 0.005-0,046) to define five different categories. Samples were then
assigned to one of the categories according to their Ct value and percentage of methylated
reference (PMR) value was calculated using the appropriate amount of 100%M from the
corresponding category (ranging from 5ng to 0.01ng). Samples with a Ct value >35 were
excluded from further analysis. The distribution of Ct values as well as the number of
samples per category is indicated in Suppl. Figure 1 and Suppl. Table 2. Samples from the
same category were run on the same plate.
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Results
Supplementary Figure 1. Ct value distribution of tumor samples (n=145) in different
categories. DNA quality of samples was assessed and classified into 4 different categories
(assigned from different amounts of input DNA of universally methylated DNA) according to
Ct values and amount of amplifiable DNA (category 1: >5ng, category 2: 5ng-0.5ng, category
3: <0.5ng-0.05ng, category 4: <0.05ng-0.01ng).
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