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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The conversion-unspecific amplification of two primer pairs, encoding a bp- and a bp-fragment located in different regions of chromosome 2, were shown to amplify genomic as well as bisulfite converted DNA Figure 7 B.
Table 1 provides an overview of the analyzed kits and their applicability to different sample types.
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.
Direct input of FFPE tissue, cell lines, fresh tissues and aspirates.
EpiTect Bisulfite Kit (48), from Qiagen – Labsave
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.
This is expected since a bisulfite treatment under harsh conditions leads to a more complete conversion on the bisufite hand but to an increased inappropriate conversion on the other hand. Shown are mean values of triplicate PCR measurements. Furthermore, the occurrence of bisulfite conversion errors  is an important parameter and should be considered carefully.
PCR measurements of each bisulfite reaction were carried in triplicate. Increasing the duration of proteinase K treatment up to 48 hours and supplementing the reaction with additional proteinase K during the lysis will help to increase the yield of highly integer DNA . The bisulfite conversion was carried out using nine different kits. Handling and user friendliness of the different kits differ significantly.
Direct input of blood plasma and serum. The CFF amplicon is free of cytosines within the sense strand and therefore allows for the amplification of bisulfite-converted and genomic DNA. Methylated cytosine exhibits a similar base pairing behaviour as cytosine and therefore methlyated and unmethylated cytosines are difficult to distinguish from each other by conventional hybridization-based molecular biological methods, i.
Hence, unconverted and converted DNA is amplified with the same efficiency. The conversion efficiency was tested by means of a PCR product generated using primers targeting cytosine-free priming CFP sites. Alternatively, high-molarity, high-temperature protocols have been developed by Hayatsu and co-workers .
Furthermore, prolonged incubation times lead to an increase of the time-to-results, therefore hampering the application of respective protocols for routine diagnostic purposes where rapid analyses are required to trigger clinical decisions. The results show a robust assay suitable for accurate DNA quantification Figure 1. In this study, the performance of nine kits was evaluated: Chr2,— and bp; GRCh These sample types each represent their specific technological challenges, i.
Table 1 List of the different kits for the preparation of bisulfite-converted DNA from various sources. InFrommer et al.
EpiTect Bisulfite Kit (48)
It should epitwct be considered that the conversion efficiency of cytosines depends on the sequence context . The CFP assay was used to quantify the specific conversion of cytosine to uracil.
Yield of bisulfite-converted DNA obtained from fresh homogenized placental tissue 0. There are no further patents, products in development or marketed products to declare. The funder had no role in study bisulfie, data collection and analysis, decision to publish, or preparation of the manuscript.
These patents are commercially exploited by Epigenomics AG. This DNA might further suffer from degradation due to storage.
Several studies describe the sensitive analysis of DNA methylation biomarkers in plasma or serum as promising tests for early detection of various tumors . Chr13,— as previously described  was used to quantify the total amount of unconverted and converted DNA simultaneously forward primer: Each bisulfite reaction was performed in triplicate. Accordingly, these eluates could be used to quantify the inhibitory effect of impurities derived from the different kit protocols.
The respective bisulfite chemistry is mainly responsible for fragmentation. However, only a few kits allow for the modification of DNA from challenging input sample material, i. J Vet Diagn Invest. In summary, high bisulfite concentrations and high temperature at prolonged incubations times will lead to a complete conversion of all cytosines to uracils on the one hand but will cause DNA degradation and inappropriate conversion of methylated cytosines to thymines on the other hand .
Hayatsu summarized the principle of the bisulfite reaction .
EpiTect Bisulfite Kit (48) from QIAGEN | SelectScience
Accordingly, slightly different results can be expected when using a different test system, i. Conventional bisulfite conversion protocols require hours of exposure to low-molarity, epitecct bisulfite.
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.
Another critical parameter is the stability of bisulfite solutions.