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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 CFF qPCR Assay.Quantitative real-time PCR analysis of a dilution series of genomic (unconverted) and bisulfite-converted DNA (10, 5, 2.5, 1.25, 0.625 ng per PCR reaction) using the CFF assay. The CFF amplicon is free of cytosines within the sense strand and therefore allows for the amplification of bisulfite-converted and genomic DNA. Shown are mean values (± standard deviation) of triplicate measurements. The assay showed a PCR efficiency of 2.0 for both templates.
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pone-0093933-g001: Analytical Performance of the CFF qPCR Assay.Quantitative real-time PCR analysis of a dilution series of genomic (unconverted) and bisulfite-converted DNA (10, 5, 2.5, 1.25, 0.625 ng per PCR reaction) using the CFF assay. The CFF amplicon is free of cytosines within the sense strand and therefore allows for the amplification of bisulfite-converted and genomic DNA. Shown are mean values (± standard deviation) of triplicate measurements. The assay showed a PCR efficiency of 2.0 for both templates.

Mentions: The DNA yield was determined by UV spectrophotometry and a quantitative real-time PCR assay measuring a cytosine free fragment (CFF). The CFF assay targets a locus containing no cytosines within the sense strand. Therefore, the sense strand of this locus is not altered during bisulfite conversion and can be used to quantify bisulfite as well as genomic DNA without the introduction of a bias. Accordingly, this assay allows for the direct comparison of genomic input DNA and bisulfite output DNA. The results obtained from bisulfite converted DNA were corrected by the factor 2 since in genomic DNA the sense and antisense of the double stranded DNA act as template whereas in bisulfite DNA only the sense strand can be amplified by PCR leading to a shift of 1 CT. The results show a robust assay suitable for accurate DNA quantification (Figure 1). Each kit was tested with high molecular weight (HMW) DNA extracted from fresh placental tissue and DNA extracted from FFPE placental tissue. The results are illustrated in Figure 2 and summarized in Table 2. In summary, all kits showed high yields of bisulfite converted DNA ranging between 29% and 92%. DNA quantification using UV showed the lowest yield when applying the EpiTect Fast FFPE and EpiTect Fast DNA Bisulfite kits. Highest DNA yields both with HMW and FFPE tissue DNA were obtained using the innuCONVERT Bisulfite kit family. The DNA yield analyzed by real time PCR showed similar results. Accordingly, these kits are of particular usability when samples are processed which are expected to contain only minute DNA amounts, i.e. microdissected cells. Again the EpiTect Fast FFPE and EpiTect Fast DNA Bisulfite kits showed the lowest DNA yield both on HMW DNA and FFPE tissue DNA. With regard to HMW DNA the EZ DNA Methylation-Gold Kit yielded the highest DNA amounts while the innuCONVERT Bisulfite kit family showed the highest yield when applying FFPE tissue DNA.


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 CFF qPCR Assay.Quantitative real-time PCR analysis of a dilution series of genomic (unconverted) and bisulfite-converted DNA (10, 5, 2.5, 1.25, 0.625 ng per PCR reaction) using the CFF assay. The CFF amplicon is free of cytosines within the sense strand and therefore allows for the amplification of bisulfite-converted and genomic DNA. Shown are mean values (± standard deviation) of triplicate measurements. The assay showed a PCR efficiency of 2.0 for both templates.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0093933-g001: Analytical Performance of the CFF qPCR Assay.Quantitative real-time PCR analysis of a dilution series of genomic (unconverted) and bisulfite-converted DNA (10, 5, 2.5, 1.25, 0.625 ng per PCR reaction) using the CFF assay. The CFF amplicon is free of cytosines within the sense strand and therefore allows for the amplification of bisulfite-converted and genomic DNA. Shown are mean values (± standard deviation) of triplicate measurements. The assay showed a PCR efficiency of 2.0 for both templates.
Mentions: The DNA yield was determined by UV spectrophotometry and a quantitative real-time PCR assay measuring a cytosine free fragment (CFF). The CFF assay targets a locus containing no cytosines within the sense strand. Therefore, the sense strand of this locus is not altered during bisulfite conversion and can be used to quantify bisulfite as well as genomic DNA without the introduction of a bias. Accordingly, this assay allows for the direct comparison of genomic input DNA and bisulfite output DNA. The results obtained from bisulfite converted DNA were corrected by the factor 2 since in genomic DNA the sense and antisense of the double stranded DNA act as template whereas in bisulfite DNA only the sense strand can be amplified by PCR leading to a shift of 1 CT. The results show a robust assay suitable for accurate DNA quantification (Figure 1). Each kit was tested with high molecular weight (HMW) DNA extracted from fresh placental tissue and DNA extracted from FFPE placental tissue. The results are illustrated in Figure 2 and summarized in Table 2. In summary, all kits showed high yields of bisulfite converted DNA ranging between 29% and 92%. DNA quantification using UV showed the lowest yield when applying the EpiTect Fast FFPE and EpiTect Fast DNA Bisulfite kits. Highest DNA yields both with HMW and FFPE tissue DNA were obtained using the innuCONVERT Bisulfite kit family. The DNA yield analyzed by real time PCR showed similar results. Accordingly, these kits are of particular usability when samples are processed which are expected to contain only minute DNA amounts, i.e. microdissected cells. Again the EpiTect Fast FFPE and EpiTect Fast DNA Bisulfite kits showed the lowest DNA yield both on HMW DNA and FFPE tissue DNA. With regard to HMW DNA the EZ DNA Methylation-Gold Kit yielded the highest DNA amounts while the innuCONVERT Bisulfite kit family showed the highest yield when applying FFPE tissue DNA.

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