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FFPEバイサルファイト変換キット

疾患状態およびエピジェネティックなプロファイルに臨床成績が関連することから、FFPEサンプルは非常に価値があります。アクティブ・モティフのFFPEバイサルファイト変換キットは、ホルマリン固定パラフィン包埋(FFPE)組織サンプルにおけるDNAメチル化分析を可能とし、単一塩基対の分解能でのDNAメチル化プロファイルを提供します。

 

Schematic of bisulfite conversion.
バイサルファイト変換の概略図

FFPEサンプルは、不適切な条件下で長期間保存される場合があります。また、組織構造を保護するために必要な強刺激の化学物質は、DNAをフラグメント化し、分解してしまいます。そこで、FFPEバイサルファイト変換キットは、DNAを最小限の分解に抑えながら、99%以上の変換効率を有する高品質なDNAを回収するために、DNA単離、精製、バイサルファイト変換、脱スルホン化およびDNA精製のための試薬が最適化されています。

 

FFPE Bisulfite Conversion Kitの特長

  • 最適化された試薬とプロトコールを用いた完全なワークフロー(バイサルファイト変換とDNA精製を介したDNA抽出)
  • 最大4 x 20 umFFPE組織切片または反応あたり35 mgの非切片コアサンプルでワーク
  • 5 pgから2 ugInput DNAにおけるバイサルファイト変換の成功
  • バイサルファイト変換が成功した場合、ヒトまたはマウスDNAだけが増幅する変換特異的PCRプライマーペアーであるポジティブコントロールの同梱

FFPEバイサルファイト変換キットは、40回のDNA単離と40回のバイサルファイト変換反応を行うことができます。FFPEバイサルファイト変換に関する他の詳細については、下にあるMethodもしくはContentsタブをクリックしてください。マニュアルまたは他の関連文書をご覧になる場合は、下のプロトコルおよび資料タブをクリックしてください。

 

 
Name Format Cat No. 価格 (税抜)  
FFPE Bisulfite Conversion Kit 40 rxns 55021 ¥95,000 Buy Now

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.

Illustration of bisulfite conversion reaction
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.

Flow Chart of the FFPE Bisulfite Conversion Kit
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.

Image of 220 bp PCR amplicon using the conversion-specific PCR primer pair with human kidney, human stomach and mouse kidney FFPE tissue
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 & 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