The Qiagen Epitect Bisulfite kit converts DNA in one step which is followed by a clean up step. Following the bisulfite conversion, I ran a PCR using the. U can try EZ Direct Methylation kit from Zymo research. For formalin fixed tissues, u need to increase the digestion time as well as volume of Proteinase K. Both. during purification. QIAGEN’s EpiTect® Fast Bisulfite kits prevent DNA fragmentation during bisulfite conversion thanks to the unique DNA. Protect Buffer, which.

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Methylation of the promoter of the MGMT gene in gliomas allows for the prediction of the response to alkylating agents [3]. Kits based on long incubation times lead to a higher fragmentation of DNA. DNA was bisulfite converted using nine different commercially available kits. Am J Transl Res.

EpiTect Bisulfite Kit (48) from QIAGEN | SelectScience

The methods for measuring bisulfite conversion reaction rates as described herein might be used in the future to identify bisklfite optimal reaction conditions allowing for sufficient conversion of cytosines but leading to only limited inappropriate conversion of methylated cytosines. The two conversion-unspecific oligonucleotides bind to genomic sites which do not contain any cytosines and therefore amplify converted, partly converted, and unconverted DNA.

Furthermore, the occurrence of bisulfite conversion errors [27][28] is an important parameter and should be considered carefully. Chr13,— as previously described [26] was used to quantify the total amount of unconverted and converted DNA simultaneously forward primer: At the same time, the kits resulted bisulrite only moderate inappropriate conversion between 0.


Introduction 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]. Water was applied to nine different bisulfite conversion kits and processed like sample DNAs process negative control sample. Several kits blsulfite bisulfite conversion of DNA are commercially available each showing advantages and disadvantages.

DNA degradation is mainly caused by depurination and depyrimidation leading to abasic sites [29][31]followed by DNA strand breaks due to N-glycoside bond cleavage. The DNA was extracted by phenol-chloroform extraction as previously described [25].

The CFP sites do not comprise any cytosines and therefore are not altered during the bisulfite conversion. Each kit was tested in nine replicates.

Accordingly, long-term storage of bisulfite-converted DNA might be possible when choosing appropriate storage buffer conditions. Hayatsu summarized the principle of the bisulfite reaction [32].

EpiTect Bisulfite Kit (48)

Increasing volumes of these process negative controls were spiked into PCR reactions each containing a constant amount of template DNA. J Vet Diagn Invest. The following kits were applied: Support Center Support Center.

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.

It should to be considered that the conversion efficiency of cytosines depends on the sequence context [27]. DNA conversion efficiencies determined in this study ranged from The aim of this study is to evaluate the performance of the most widely used kits: In this study, the performance of nine kits was evaluated: Table 1 provides an overview of the analyzed kits and their applicability to different sample types.


This article has been cited by other articles in PMC. 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. The DNA from FFPE tissue already showed a strong fragmentation before bisulfite conversion, therefore only minor differences between the kits can be observed after conversion.

There is an emerging need for protocols and kits that allow for the preparation of bisulfite DNA from low abundance biomarkers in high volume body fluidic samples of clinical relevance, i.

The analytical performance of the assay was characterized by measuring mixtures of methylated and unmethylated DNA Figure 3. Bisulfite conversion and subsequent purification was performed according to the respective kit protocols.

The bisulfite conversion was carried out using nine different kits.

EpiTect 96 Bisulfite Kit (2) from QIAGEN | SelectScience

Direct input of FFPE tissue sections, cultured cell lines, fresh tissues and whole blood. These figures are in concordance with a previous study, which reported inappropriate conversion rates between 0.

However, several technological advances have now led to protocols which are much more convenient and user friendly compared to the original 16 hours protocol [19][20][21][22][23][24]. These eluates process negative control samples were expected to contain similar impurities as Epotect samples processed with the respective kits.