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Tools for translational epigenetic studies involving formalin-fixed paraffin-embedded human tissue: applying the Infinium HumanMethyation450 Beadchip assay to large population-based studies.

Wong EM, Joo JE, McLean CA, Baglietto L, English DR, Severi G, Hopper JL, Milne RL, FitzGerald LM, Giles GG, Southey MC - BMC Res Notes (2015)

Bottom Line: Five samples did not have a detectable amount of DNA with an additional 42 failing to progress past QC checkpoint 3.The modified protocol is suitable for the analysis of FFPE tumour-enriched DNA and can be systematically applied to hundreds of samples.This protocol will have utility in population-based translational epigenetic studies and is applicable to a wide variety of translated studies interested in analysis of methylation and its role in the predisposition to disease and disease progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Melbourne, Melbourne, VIC, 3010, Australia. emwong@unimelb.edu.au.

ABSTRACT

Background: Large population-based translational epigenetic studies are emerging due to recent technological advances that have made molecular analyses possible. For example, the Infinium HumanMethylation450 Beadchip (HM450K) has enabled studies of genome-wide methylation on a scale not previously possible. However, application of the HM450K to DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumour material has been more challenging than application to high quality DNA extracted from blood. To facilitate the application of this assay consistently across a large number of FFPE tumour-enriched DNA samples we have devised a modification to the HM450K protocol for FFPE that includes an additional quality control (QC) checkpoint.

Results: QC checkpoint 3 was designed to assess the presence of DNA after bisulfite conversion and restoration, just prior to application of the HM450K assay. DNA was extracted from 474 archival FFPE breast tumour material. Five samples did not have a detectable amount of DNA with an additional 42 failing to progress past QC checkpoint 3. Genome-wide methylation was measured for the remaining 428 tumour-enriched DNA. Of these, only 4 samples failed our stringent HM450K data criteria thus representing a 99% success rate. Using prior knowledge about methylation marks associated with breast cancer we further explored the quality of the data. Twenty probes in the BRCA1 promoter region showed increased methylation in triple-negative breast cancers compared to Luminal A, Luminal B and HER2-positive breast cancer subtypes. Validation of this observation in published data from The Cancer Genome Atlas (TCGA) Network (obtained from DNA extracted from fresh frozen tumour samples) confirms the quality of the data obtained from the improved protocol.

Conclusions: The modified protocol is suitable for the analysis of FFPE tumour-enriched DNA and can be systematically applied to hundreds of samples. This protocol will have utility in population-based translational epigenetic studies and is applicable to a wide variety of translated studies interested in analysis of methylation and its role in the predisposition to disease and disease progression.

No MeSH data available.


Related in: MedlinePlus

Workflow diagram for assessing the suitability of FFPE tumour-enriched DNA on the HM450K platform. A novel checkpoint (QC checkpoint 3) has been incorporated in addition to the standard protocol to assess DNA availability after sodium bisulfite modification and restoration. The number of samples entering and exiting each QC checkpoint is as indicated
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Fig1: Workflow diagram for assessing the suitability of FFPE tumour-enriched DNA on the HM450K platform. A novel checkpoint (QC checkpoint 3) has been incorporated in addition to the standard protocol to assess DNA availability after sodium bisulfite modification and restoration. The number of samples entering and exiting each QC checkpoint is as indicated

Mentions: To address these issues, we adopted a modified protocol (Fig. 1) based on the recommended Illumina Infinium workflow for FFPE tumour-enriched DNA which incorporated a third quality control (QC) checkpoint in the form of an in-house designed bisulfite-specific qPCR assay to assess the capacity of the protocol to support large population-based translation epigenetics studies.Fig. 1


Tools for translational epigenetic studies involving formalin-fixed paraffin-embedded human tissue: applying the Infinium HumanMethyation450 Beadchip assay to large population-based studies.

Wong EM, Joo JE, McLean CA, Baglietto L, English DR, Severi G, Hopper JL, Milne RL, FitzGerald LM, Giles GG, Southey MC - BMC Res Notes (2015)

Workflow diagram for assessing the suitability of FFPE tumour-enriched DNA on the HM450K platform. A novel checkpoint (QC checkpoint 3) has been incorporated in addition to the standard protocol to assess DNA availability after sodium bisulfite modification and restoration. The number of samples entering and exiting each QC checkpoint is as indicated
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4595238&req=5

Fig1: Workflow diagram for assessing the suitability of FFPE tumour-enriched DNA on the HM450K platform. A novel checkpoint (QC checkpoint 3) has been incorporated in addition to the standard protocol to assess DNA availability after sodium bisulfite modification and restoration. The number of samples entering and exiting each QC checkpoint is as indicated
Mentions: To address these issues, we adopted a modified protocol (Fig. 1) based on the recommended Illumina Infinium workflow for FFPE tumour-enriched DNA which incorporated a third quality control (QC) checkpoint in the form of an in-house designed bisulfite-specific qPCR assay to assess the capacity of the protocol to support large population-based translation epigenetics studies.Fig. 1

Bottom Line: Five samples did not have a detectable amount of DNA with an additional 42 failing to progress past QC checkpoint 3.The modified protocol is suitable for the analysis of FFPE tumour-enriched DNA and can be systematically applied to hundreds of samples.This protocol will have utility in population-based translational epigenetic studies and is applicable to a wide variety of translated studies interested in analysis of methylation and its role in the predisposition to disease and disease progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Melbourne, Melbourne, VIC, 3010, Australia. emwong@unimelb.edu.au.

ABSTRACT

Background: Large population-based translational epigenetic studies are emerging due to recent technological advances that have made molecular analyses possible. For example, the Infinium HumanMethylation450 Beadchip (HM450K) has enabled studies of genome-wide methylation on a scale not previously possible. However, application of the HM450K to DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumour material has been more challenging than application to high quality DNA extracted from blood. To facilitate the application of this assay consistently across a large number of FFPE tumour-enriched DNA samples we have devised a modification to the HM450K protocol for FFPE that includes an additional quality control (QC) checkpoint.

Results: QC checkpoint 3 was designed to assess the presence of DNA after bisulfite conversion and restoration, just prior to application of the HM450K assay. DNA was extracted from 474 archival FFPE breast tumour material. Five samples did not have a detectable amount of DNA with an additional 42 failing to progress past QC checkpoint 3. Genome-wide methylation was measured for the remaining 428 tumour-enriched DNA. Of these, only 4 samples failed our stringent HM450K data criteria thus representing a 99% success rate. Using prior knowledge about methylation marks associated with breast cancer we further explored the quality of the data. Twenty probes in the BRCA1 promoter region showed increased methylation in triple-negative breast cancers compared to Luminal A, Luminal B and HER2-positive breast cancer subtypes. Validation of this observation in published data from The Cancer Genome Atlas (TCGA) Network (obtained from DNA extracted from fresh frozen tumour samples) confirms the quality of the data obtained from the improved protocol.

Conclusions: The modified protocol is suitable for the analysis of FFPE tumour-enriched DNA and can be systematically applied to hundreds of samples. This protocol will have utility in population-based translational epigenetic studies and is applicable to a wide variety of translated studies interested in analysis of methylation and its role in the predisposition to disease and disease progression.

No MeSH data available.


Related in: MedlinePlus