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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.


Experimental overview of the investigated sources of variability.Roman numerals represent the investigated sources of variability. Left side: default parameters, marked by an asterisk (*). Right side: alternative parameters. Alternative parameters were tested individually with two exceptions; first, both the default and the alternative input amount in the bisulfite conversion (III) were tested with both bisulfite kits (IV), and second, for testing of storage (V), DNA was bisulfite converted in parallel with the two kits. For Zymo calculations, 500 ng input was used as default parameter. Abbreviations: PMR, percent of methylated reference; qMSP, quantitative methylation-specific PCR.
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f1: Experimental overview of the investigated sources of variability.Roman numerals represent the investigated sources of variability. Left side: default parameters, marked by an asterisk (*). Right side: alternative parameters. Alternative parameters were tested individually with two exceptions; first, both the default and the alternative input amount in the bisulfite conversion (III) were tested with both bisulfite kits (IV), and second, for testing of storage (V), DNA was bisulfite converted in parallel with the two kits. For Zymo calculations, 500 ng input was used as default parameter. Abbreviations: PMR, percent of methylated reference; qMSP, quantitative methylation-specific PCR.

Mentions: In the present study, qMSP is the representative method for investigating potential sources of variability in bisulfite PCR-based methylation analyses. Figure 1 illustrates the individual sources of variability (marked with roman numerals) that have been tested, including; I) investigator, II) time point of analysis, III) DNA input amount in the bisulfite conversion, IV) bisulfite conversion kit, V) storage (of bisulfite converted DNA), VI) template amount in the qMSP, and VII) reference for normalization. For each source of variability, one or more alternative parameter(s) were tested against a default (marked with an asterisk in Fig. 1). Alternative parameters were tested individually: when an alternative parameter was chosen for one given source of variability, default parameters were used for the rest of the pipeline (exceptions are indicated in the legend of Fig. 1).


Experimental factors affecting the robustness of DNA methylation analysis
Experimental overview of the investigated sources of variability.Roman numerals represent the investigated sources of variability. Left side: default parameters, marked by an asterisk (*). Right side: alternative parameters. Alternative parameters were tested individually with two exceptions; first, both the default and the alternative input amount in the bisulfite conversion (III) were tested with both bisulfite kits (IV), and second, for testing of storage (V), DNA was bisulfite converted in parallel with the two kits. For Zymo calculations, 500 ng input was used as default parameter. Abbreviations: PMR, percent of methylated reference; qMSP, quantitative methylation-specific PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Experimental overview of the investigated sources of variability.Roman numerals represent the investigated sources of variability. Left side: default parameters, marked by an asterisk (*). Right side: alternative parameters. Alternative parameters were tested individually with two exceptions; first, both the default and the alternative input amount in the bisulfite conversion (III) were tested with both bisulfite kits (IV), and second, for testing of storage (V), DNA was bisulfite converted in parallel with the two kits. For Zymo calculations, 500 ng input was used as default parameter. Abbreviations: PMR, percent of methylated reference; qMSP, quantitative methylation-specific PCR.
Mentions: In the present study, qMSP is the representative method for investigating potential sources of variability in bisulfite PCR-based methylation analyses. Figure 1 illustrates the individual sources of variability (marked with roman numerals) that have been tested, including; I) investigator, II) time point of analysis, III) DNA input amount in the bisulfite conversion, IV) bisulfite conversion kit, V) storage (of bisulfite converted DNA), VI) template amount in the qMSP, and VII) reference for normalization. For each source of variability, one or more alternative parameter(s) were tested against a default (marked with an asterisk in Fig. 1). Alternative parameters were tested individually: when an alternative parameter was chosen for one given source of variability, default parameters were used for the rest of the pipeline (exceptions are indicated in the legend of Fig. 1).

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.