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

Date Published: April 3, 2014

Publisher: Public Library of Science

Author(s): Emily Eva Holmes, Maria Jung, Sebastian Meller, Annette Leisse, Verena Sailer, Julie Zech, Martina Mengdehl, Leif-Alexander Garbe, Barbara Uhl, Glen Kristiansen, Dimo Dietrich, Zhuang Zuo.


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.

Partial Text

DNA methylation of cytosines within the CpG dinucleotide context is an epigenetic mechanism, which plays an important role in biological processes, such as cell differentiation and development [1]. Furthermore, aberrant DNA methylation is a hallmark of malignant tumors and plays a key role during carcinogenesis [2]. Studies on DNA methylation changes in the course of cancer development and progression will broaden the understanding of this devastating disease and will lead to several clinically relevant biomarkers and therapy approaches in the future. A few DNA methylation biomarkers are already on the road to clinical use for predictive, diagnostic and screening purposes. Methylation of the promoter of the MGMT gene in gliomas allows for the prediction of the response to alkylating agents [3]. The MGMT promoter methylation status has become a parameter for stratification of patients with glioma within several clinical trials [4]. Macrodissected tumor tissues from sections of FFPE tumors are the sample of choice to achieve good results [4]. Two additional tests based on the methylation analysis in FFPE tissues already show a high level of validation qualifying them for clinical use. The ConfirmMDx test (MDxHealth, Inc., Irvine, CA, USA) is based on DNA methylation of RASSF1, APC, and GSTP1 in FFPE biopsies [5] and intends to help distinguish patients who have a true negative biopsy from patients who may have occult prostate cancer. DNA methylation of PITX2 in FFPE prostatectomy specimens is a strong prognostic biomarker for identifying patients who are at high risk to suffer from prostate-specific antigen (PSA) recurrence after radical ectomy [6], [7], [8], [9]. Free-circulating methylated SEPT9 gene copies in plasma as a screening biomarker for colorectal cancer were recently validated in a large observational prospective screening trial including more than 7,000 asymptomatic subjects [10]. SHOX2 DNA methylation is another plasma based biomarker that allows for the identification of lung cancer [11]. Furthermore, SHOX2 DNA methylation is a validated biomarker for detecting lung cancer in the cellular fraction of bronchial aspirates [12], [13] and pleural effusions [14], [15] as well as in EBUS-TBNA (endobronchial ultrasound with transbronchial needle aspiration) specimens [16]. These examples of methylation biomarkers with the highest level of validation clearly indicate the necessity of technologies, which allow for the accurate determination of DNA methylation in various sample types. These sample types each represent their specific technological challenges, i.e. DNA fragmentation in FFPE tissues and low abundance of methylated copies in blood plasma. The availability of kits and tools to measure DNA methylation in these sample types is mandatory to open this research area to a wide group of researchers.

Several kits for bisulfite conversion of DNA are commercially available each showing advantages and disadvantages. The choice of a suitable kit for a specific application is not trivial and should be based on the specific performance requirements with regard to the respective sample material and biological/clinical question. While hands-on-time and time to result are usually rather an issue for routine molecular diagnostics, overall high yield of DNA suitable for downstream molecular analyses is mandatory in the fields of research and diagnostics. Furthermore, the occurrence of bisulfite conversion errors [27], [28] is an important parameter and should be considered carefully.