FFPE Bisulfite Conversion Kit

Synonyms

DNA methylation, 5 mC, 5mC, FFPE, formalin-fixed paraffin embedded, clinical sample, bisulfite, bisulphite, methyl, bisulfite conversion, sodium bisulfite, methylated DNA, CpG, cytosine, uracil, conversion

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Overview

FFPE samples are highly valuable for their ability to relate clinical outcomes to disease states and epigenetic profiles. Active Motif's FFPE Bisulfite Conversion Kit is designed for DNA methylation analysis of formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues to provide a DNA methylation profile at single-base-pair resolution.

Since FFPE samples are often stored for long periods of time under less than optimal conditions and the harsh chemicals needed to preserve tissue structure often lead to highly fragmented and degraded DNA, the FFPE Bisulfite Conversion Kit provides optimized reagents for DNA isolation, purification, bisulfite conversion, desulphonation and a final DNA purification to improve recovery of high quality DNA with minimal degradation and greater than 99% conversion efficiency.

The FFPE Bisulfite Conversion Kit contains enough reagents to perform 40 DNA isolation and 40 bisulfite conversion reactions. To learn more about FFPE Bisulfite Conversion, click on the Method, or Contents tabs. To view a manual or other related documents, click on the Documents tab.

Method

How does the FFPE Bisulfite Conversion Kit work?

Bisulfite conversion followed by DNA sequencing has long been considered the "gold standard" in site-specific DNA methylation analysis as it provides single-base-pair resolution of the DNA methylation profile. The conversion reaction occurs as a 3-step deamination of cytosine residues into uracil. As only unmethylated cytosine residues are susceptible to bisulfite conversion, the original methylation state of the DNA can be determined.

Figure 1: Schematic of the bisulfite conversion reaction of unmethylated cytosine residues to uracil.

The first step in the bisulfite conversion reaction is sulfonation, where sodium bisulfite is added to the double bond of cytosine to form a cytosine-bisulfite derivative. This reaction is reversible, with the forward reaction being favored by high temperature and low pH. The second step is an irreversible hydrolytic deamination of the cytosine-bisulfite derivative that results in uracil-bisulfite derivative. This reaction is also favored by low pH. The final step involves desulfonation of the uracil-bisulfite to uracil under high pH conditions. Only unmethylated cytosines are susceptible to the bisulfite reaction. Methylated cytosines do not undergo conversion.

In the FFPE Bisulfite Conversion Kit, first, DNA is extracted from formalin- or paraformalin-fixed, paraffin-embedded tissue sections or unsectioned core samples. Then, DNA is heated to reverse protein-DNA cross-links caused by fixation. Samples are then treated with a protease solution to degrade proteins and the DNA is column purified prior to bisulfite conversion. Finally, Active Motif's optimized bisulfite conversion reagents and protocol are used to convert cytosine residues not protected by methylation into uracils to provide a DNA methylation profile at single-base-pair resolution.

Figure 2: Flow Chart of the FFPE Bisulfite Conversion Kit.

Also included in the FFPE Bisulfite Conversion Kit is a positive control conversion-specific PCR primer pair that is specific for human or mouse DNA. The primer pair will produce a 220 bp PCR amplicon only if bisulfite conversion was successful. This enable validation of bisulfite conversion success prior to downstream sequencing.

Figure 3: Schematic of the bisulfite conversion reaction of unmethylated cytosine residues to uracil.

Active Motif's FFPE Bisulfite Conversion Kit was used to isolate genomic DNA from human kidney, human stomach and mouse kidney FFPE tissue sections. Bisulfite conversion was performed using the input DNA quantities listed and converted DNA was PCR amplified using the included conversion-specific PCR primer pair. Results show a 220 bp PCR amplicon that is present only with bisulfite treated samples and indicates that as little as 5 pg DNA can be used for successful bisulfite conversion.

Contents

Contents & Storage

The FFPE Bisulfite Conversion Kit is shipped on dry ice but contains items with multiple storage temperatures. We recommend storing each component at the temperatures listed in the table below. Components are guaranteed stable for 6 months from the date of receipt when stored properly. This FFPE Bisulfite Conversion Kit includes the following components:

• Protease Solution; Store at -20°C
• RNase A; Store at -20°C
• Glycogen; Store at -20°C
• Conversion-specific PCR primer pair (4 µM); Store at -20°C
• Digestion Buffer AM3; Store at RT
• Wash Buffer 1; Store at RT
• Wash Buffer 2/3; Store at RT
• Collection tubes (bag of 40); Store at RT
• Filter Cartridges (2 bags of 20); Store at RT
• Isolation Additive; Store at RT
• Elution Buffer AM5; Store at RT
• Conversion Reagent (4 tubes, lyophilized); Store at RT
• Hydroquinone (4 tubes); Store at RT
• Buffer A; Store at RT
• Buffer B; Store at RT
• DNA Binding Buffer (2 bottles); Store at RT
• DNA Wash Buffer; Store at RT
• DNA Elution Buffer; Store at RT
• DNA purification columns and collection tubes (40 each); Store at RT

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