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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However, even though the performance differences between the kits regarding specific and inappropriate conversion are statistically significant, these differences seem not to be high enough to impair the downstream analysis of bisulfite DNA prepared with either kit.
The conversion efficiency was tested by means of a PCR product generated using primers targeting cytosine-free priming CFP sites. Accordingly, these eluates could be used to quantify the inhibitory effect of impurities derived from the different kit protocols.
EpiTect Bisulfite Kit (48)
Correlation between conversion efficiency and inappropriate conversion was bisulfute using Pearson correlation. Several kits for bisulfite conversion of DNA are commercially available each showing advantages and disadvantages. 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.
In brief, bisulfite reacts with cytosines in a single stranded DNA molecule through the formation of a 5,6-dihydrocytosinesulfonate intermediate preferably at acidic pH around pH 5. Consequently, the epigenetic information of the DNA is bisuldite into sequence information, which can be studied via PCR and other hybridization based methods.
Water was applied to nine different bisulfite conversion kits and processed like sample DNAs process negative control sample.
This article has been cited by other articles in PMC. Accordingly, a bias toward the amplification of completely converted DNA and therefore an kif of the conversion efficiency is avoided.
The bisulfite conversion is a chemical reaction under harsh chemical conditions low pH, high temperature and elongated incubation times causing significant DNA degradation .
EpiTect Bisulfite Kit (48), from Qiagen – Labsave
Informed consent written was obtained from all donors. Open in a separate window. The conversion efficiency of each kit was tested by means of clone sequencing of a PCR product generated using primers targeting cytosine-free priming CFP sites Figure 7 A.
Alternatively, high-molarity, high-temperature protocols have been developed by Hayatsu and co-workers . Furthermore, aberrant DNA methylation is a hallmark of malignant tumors and plays a key role during carcinogenesis .
EpiTect 96 Bisulfite Kit (2) from QIAGEN | SelectScience
All nine kits studied in this series of experiments showed significantly different but comparable results and high performance when applying high molecular weight HMW DNA. Kut 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. Conventional bisulfite conversion protocols require hours of exposure to low-molarity, low-temperature bisulfite.
However, several technological advances have now led to protocols which are much more convenient and user friendly compared to the original 16 hours protocol  biuslfite,  epiitect, . Table 1 provides an overview of the analyzed kits and their applicability to different sample types.
The kits led to an inappropriate conversion between 0. Phys Chem Chem Phys. The DNA was extracted by phenol-chloroform extraction as previously described .
The assay showed a PCR efficiency of 2. Author information Article notes Copyright and License information Disclaimer. Body fluids often only contain low concentrations of DNA which needs to be concentrated bisulfitr to bisulfite conversion in order to reduce the volume therefore enabling the input into the bisulfite reaction.
Curr Protoc Nucleic Acid Chem. The methods for measuring bisulfite conversion reaction rates as described herein might be used in the future to identify the optimal reaction conditions allowing for sufficient conversion of cytosines but leading to only limited inappropriate conversion of methylated cytosines.
Bisulfite-converted DNA is mainly single-stranded DNA generated under harsh chemical reaction bieulfite which cause significant degradation. The inappropriate conversion of methyl-cytosine to thymine was determined for nine different commercially available bisulfite conversion kits. Nucleic Acids Symp Ser Oxf. Furthermore, the occurrence of bisulfite conversion errors  is an important parameter and should be considered carefully. Accordingly, these kits are of particular usability when samples are processed which are expected to contain only minute DNA amounts, i.
DNA was bisulfite converted using nine different commercially available kits. National Center for Biotechnology InformationU. Each bisulfite reaction was performed in triplicate. However, only a few kits allow for the modification of DNA from challenging input sample material, i.