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Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine.

Holmes EE, Jung M, Meller S, Leisse A, Sailer V, Zech J, Mengdehl M, Garbe LA, Uhl B, Kristiansen G, Dietrich D - PLoS ONE (2014)

Bottom Line: The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis.Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit).Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits).

View Article: PubMed Central - PubMed

Affiliation: University Hospital Bonn (UKB), Institute of Pathology, Bonn, Germany.

ABSTRACT
DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine.

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Analytical Performance of the ACTB/SHOX2/SEPT9 Triplex Assay.Bisulfite-specific and methylation-specific quantitative real-time PCR analysis of mixtures of bisulfite-converted artificially methylated DNA and unmethylated DNA from sperm. The ACTB/SHOX2/SEPT9 assay amplifies bisulfite-converted methylated SHOX2 and SEPT9 gene copies and total bisulfite-converted DNA using an ACTB amplicon. Shown are mean values (± standard deviation) of triplicate measurements.
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pone-0093933-g003: Analytical Performance of the ACTB/SHOX2/SEPT9 Triplex Assay.Bisulfite-specific and methylation-specific quantitative real-time PCR analysis of mixtures of bisulfite-converted artificially methylated DNA and unmethylated DNA from sperm. The ACTB/SHOX2/SEPT9 assay amplifies bisulfite-converted methylated SHOX2 and SEPT9 gene copies and total bisulfite-converted DNA using an ACTB amplicon. Shown are mean values (± standard deviation) of triplicate measurements.

Mentions: Furthermore, a quantitative and sensitive methylation-specific triplex real-time PCR assay for determination of SHOX2 and SEPT9 DNA methylation was used to evaluate the kit performance. The determined SHOX2 and SEPT9 DNA methylation was referred to total DNA quantified using an ACTB assay. The analytical performance of the assay was characterized by measuring mixtures of methylated and unmethylated DNA (Figure 3). The ACTB assays showed the same CT value throughout the series of DNA mixtures as the applied ACTB primers are methylation-unspecific. In contrast, the SHOX2 and SEPT9 primers are methylation-specific and therefore amplify only methylated DNA. Accordingly, the CT values correlate with the relative amount of methylated DNA in the mixtures (Figure 3). Each kit was tested with high molecular weight (HMW) DNA extracted from fresh placental tissue and DNA extracted from FFPE placental tissue. Lowest yields of bisulfite converted DNA were obtained using the EpiTect Fast FFPE and EpiTect Fast DNA Bisulfite kits. Highest yield of bisulfite converted HMW DNA was obtained using the EZ DNA Methylation-Gold kit. The innuCONVERT kit family showed the highest yield when applying DNA from FFPE tissue (Figures 2 and 4, Table 2).


Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine.

Holmes EE, Jung M, Meller S, Leisse A, Sailer V, Zech J, Mengdehl M, Garbe LA, Uhl B, Kristiansen G, Dietrich D - PLoS ONE (2014)

Analytical Performance of the ACTB/SHOX2/SEPT9 Triplex Assay.Bisulfite-specific and methylation-specific quantitative real-time PCR analysis of mixtures of bisulfite-converted artificially methylated DNA and unmethylated DNA from sperm. The ACTB/SHOX2/SEPT9 assay amplifies bisulfite-converted methylated SHOX2 and SEPT9 gene copies and total bisulfite-converted DNA using an ACTB amplicon. Shown are mean values (± standard deviation) of triplicate measurements.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3974851&req=5

pone-0093933-g003: Analytical Performance of the ACTB/SHOX2/SEPT9 Triplex Assay.Bisulfite-specific and methylation-specific quantitative real-time PCR analysis of mixtures of bisulfite-converted artificially methylated DNA and unmethylated DNA from sperm. The ACTB/SHOX2/SEPT9 assay amplifies bisulfite-converted methylated SHOX2 and SEPT9 gene copies and total bisulfite-converted DNA using an ACTB amplicon. Shown are mean values (± standard deviation) of triplicate measurements.
Mentions: Furthermore, a quantitative and sensitive methylation-specific triplex real-time PCR assay for determination of SHOX2 and SEPT9 DNA methylation was used to evaluate the kit performance. The determined SHOX2 and SEPT9 DNA methylation was referred to total DNA quantified using an ACTB assay. The analytical performance of the assay was characterized by measuring mixtures of methylated and unmethylated DNA (Figure 3). The ACTB assays showed the same CT value throughout the series of DNA mixtures as the applied ACTB primers are methylation-unspecific. In contrast, the SHOX2 and SEPT9 primers are methylation-specific and therefore amplify only methylated DNA. Accordingly, the CT values correlate with the relative amount of methylated DNA in the mixtures (Figure 3). Each kit was tested with high molecular weight (HMW) DNA extracted from fresh placental tissue and DNA extracted from FFPE placental tissue. Lowest yields of bisulfite converted DNA were obtained using the EpiTect Fast FFPE and EpiTect Fast DNA Bisulfite kits. Highest yield of bisulfite converted HMW DNA was obtained using the EZ DNA Methylation-Gold kit. The innuCONVERT kit family showed the highest yield when applying DNA from FFPE tissue (Figures 2 and 4, Table 2).

Bottom Line: The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis.Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit).Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits).

View Article: PubMed Central - PubMed

Affiliation: University Hospital Bonn (UKB), Institute of Pathology, Bonn, Germany.

ABSTRACT
DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine.

Show MeSH
Related in: MedlinePlus