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Experimental factors affecting the robustness of DNA methylation analysis

View Article: PubMed Central - PubMed

ABSTRACT

Diverging methylation frequencies are often reported for the same locus in the same disease, underscoring the need for limiting technical variability in DNA methylation analyses. We have investigated seven likely sources of variability at different steps of bisulfite PCR-based DNA methylation analyses using a fully automated quantitative methylation-specific PCR setup of six gene promoters across 20 colon cancer cell lines. Based on >15,000 individual PCRs, all tested parameters affected the normalized percent of methylated reference (PMR) differences, with a fourfold varying magnitude. Additionally, large variations were observed across the six genes analyzed. The highest variation was seen using single-copy genes as reference for normalization, followed by different amounts of template in the PCR, different amounts of DNA in the bisulfite reaction, and storage of bisulfite converted samples. Finally, when a highly standardized pipeline was repeated, the difference in PMR value for the same assay in the same cell line was on average limited to five (on a 0–100 scale). In conclusion, a standardized pipeline is essential for consistent methylation results, where parameters are kept constant for all samples. Nevertheless, a certain level of variation in methylation values must be expected, underscoring the need for careful interpretation of data.

No MeSH data available.


The sum of normalized PMR difference varies substantially between gene assays.The plot illustrates the sum of normalized PMR differences (using absolute values) for all cell lines per assay, and is shown for all the tested parameters. The colours symbolize the individual assays, as indicated on top. The area of each bubble represents the sum of the absolute normalized PMR difference for the respective assay, multiplied by a factor of 25 for improved visualization. The roman numerals correspond to those in Figs 1 and 2.
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f3: The sum of normalized PMR difference varies substantially between gene assays.The plot illustrates the sum of normalized PMR differences (using absolute values) for all cell lines per assay, and is shown for all the tested parameters. The colours symbolize the individual assays, as indicated on top. The area of each bubble represents the sum of the absolute normalized PMR difference for the respective assay, multiplied by a factor of 25 for improved visualization. The roman numerals correspond to those in Figs 1 and 2.

Mentions: Interestingly, the sum of the normalized PMR differences for all cell lines (absolute values) varied substantially from gene promoter to gene promoter for several of the tested parameters (Fig. 3). SFRP1 exhibited significantly lower normalized PMR differences across all tested parameters than expected by chance (P = 0.017), while MGMT tended to have higher normalized PMR differences (P = 0.048; Supplementary Table S2). Moreover, for the individual experimental parameters, the normalized PMR differences per gene assay varied with a magnitude of more than five (Supplementary Table S3). In this regard, the lowest coefficient of variation (CV) was observed for ‘6 months storage in freezer’ (CV = 0.107), and the highest for ‘25% template in qMSP’ (CV = 0.587).


Experimental factors affecting the robustness of DNA methylation analysis
The sum of normalized PMR difference varies substantially between gene assays.The plot illustrates the sum of normalized PMR differences (using absolute values) for all cell lines per assay, and is shown for all the tested parameters. The colours symbolize the individual assays, as indicated on top. The area of each bubble represents the sum of the absolute normalized PMR difference for the respective assay, multiplied by a factor of 25 for improved visualization. The roman numerals correspond to those in Figs 1 and 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037394&req=5

f3: The sum of normalized PMR difference varies substantially between gene assays.The plot illustrates the sum of normalized PMR differences (using absolute values) for all cell lines per assay, and is shown for all the tested parameters. The colours symbolize the individual assays, as indicated on top. The area of each bubble represents the sum of the absolute normalized PMR difference for the respective assay, multiplied by a factor of 25 for improved visualization. The roman numerals correspond to those in Figs 1 and 2.
Mentions: Interestingly, the sum of the normalized PMR differences for all cell lines (absolute values) varied substantially from gene promoter to gene promoter for several of the tested parameters (Fig. 3). SFRP1 exhibited significantly lower normalized PMR differences across all tested parameters than expected by chance (P = 0.017), while MGMT tended to have higher normalized PMR differences (P = 0.048; Supplementary Table S2). Moreover, for the individual experimental parameters, the normalized PMR differences per gene assay varied with a magnitude of more than five (Supplementary Table S3). In this regard, the lowest coefficient of variation (CV) was observed for ‘6 months storage in freezer’ (CV = 0.107), and the highest for ‘25% template in qMSP’ (CV = 0.587).

View Article: PubMed Central - PubMed

ABSTRACT

Diverging methylation frequencies are often reported for the same locus in the same disease, underscoring the need for limiting technical variability in DNA methylation analyses. We have investigated seven likely sources of variability at different steps of bisulfite PCR-based DNA methylation analyses using a fully automated quantitative methylation-specific PCR setup of six gene promoters across 20 colon cancer cell lines. Based on >15,000 individual PCRs, all tested parameters affected the normalized percent of methylated reference (PMR) differences, with a fourfold varying magnitude. Additionally, large variations were observed across the six genes analyzed. The highest variation was seen using single-copy genes as reference for normalization, followed by different amounts of template in the PCR, different amounts of DNA in the bisulfite reaction, and storage of bisulfite converted samples. Finally, when a highly standardized pipeline was repeated, the difference in PMR value for the same assay in the same cell line was on average limited to five (on a 0–100 scale). In conclusion, a standardized pipeline is essential for consistent methylation results, where parameters are kept constant for all samples. Nevertheless, a certain level of variation in methylation values must be expected, underscoring the need for careful interpretation of data.

No MeSH data available.