Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

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.

SOCS3 Modulates the Response to Enzalutamide and Is Regulated by Androgen Receptor Signaling and CpG Methylation in Prostate Cancer Cells

“…Briefly, DNA was isolated from formalin-fixed, paraffin-embedded (FFPE) tissue blocks and subsequently converted with bisulfite using the InnuCONVERT Bisulfite All-In-One Kit (Analytik Jena) as previously described (27). Quantification of methylated DNA was performed by duplex qPCR using methylation-specific primers for SOCS3 and methylation-insensitive primers for ACTB as a reference (Table 1).…”

Methylation analyses in liquid biopsy

“…Increased recovery is mostly attained by reducing the incubation time of DNA with bisulfite conversion reagent. Comparisons of the main features of some of these kits with regards to DNA recovery, DNA fragmentation and conversion efficiency were recently published (60,61). Again, due to the lack of standardization of the quantification methods, DNA yield is inconsistent across studies.…”

Current status and future perspectives of circulating cell-free DNA methylation in clinical diagnostics

“…urine, ascites, pleural effusions, can be used for the analysis of methylated ccfDNA as well. Each sample type exhibits its own challenges with regard to the analysis of methylated ccfDNA [41][42][43]. However, all sample types share one feature which mainly dictates the choice of methods for the next preanalytical steps: Disease-derived ccfDNA in clinically relevant samples is usually low abundant.…”

“…Accordingly, the preanalytical workflow starts with a concentration of the samples by means of magnetic bead-based DNA extraction or a polymer-mediated enrichment [42,43]. Most methodologies for DNA methylation analyses require a preceding treatment of the DNA with bisulfite.…”

“…During bisulfite treatment, cytosines are deaminated and converted into uracils while methylated cytosines remain mainly unaffected [44]. The bisulfite conversion is a harsh chemical reaction at elevated temperatures, prolonged incubation times and low pH leading to a significant degradation of DNA and an unspecific conversion of methylated cytosines Brought to you by | MIT Libraries Authenticated Download Date | 5/10/18 10:54 AM [42]. The DNA extraction/ concentration, followed by the bisulfite reaction and another subsequent DNA purification is laboratory intensive, significantly influencing the time-to-result and the overall costs, including labor.…”