Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine.
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.
University Hospital Bonn (UKB), Institute of Pathology, Bonn, Germany.
Emily Eva
EE
Holmes
University Hospital Bonn (UKB), Institute of Pathology, Bonn, Germany.
Maria
M
Jung
University Hospital Bonn (UKB), Institute of Pathology, Bonn, Germany.
Sebastian
S
Meller
University Hospital Bonn (UKB), Institute of Pathology, Bonn, Germany.
Annette
A
Leisse
University Hospital Bonn (UKB), Institute of Pathology, Bonn, Germany.
Verena
V
Sailer
Berlin Institute of Technology (TU Berlin), Biotechnology, Berlin, Germany.
Julie
J
Zech
Berlin Institute of Technology (TU Berlin), Biotechnology, Berlin, Germany.
Martina
M
Mengdehl
Berlin Institute of Technology (TU Berlin), Biotechnology, Berlin, Germany.
Leif-Alexander
LA
Garbe
University Hospital Bonn (UKB), Institute of Pathology, Bonn, Germany.
Barbara
B
Uhl
University Hospital Bonn (UKB), Institute of Pathology, Bonn, Germany.
Glen
G
Kristiansen
University Hospital Bonn (UKB), Institute of Pathology, Bonn, Germany.
Dimo
D
Dietrich
D001826:Body Fluids / Q000737:chemistry*; D002460:Cell Line / Q000737:chemistry*; D004247:DNA / Q000032:analysis*; D019175:DNA Methylation; D006801:Humans; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D013447:Sulfites
D011933:Reagent Kits, Diagnostic; D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite