EpiTect Fast Bisulfite Conversion Kits

For ultrafast, highly efficient bisulfite conversion from gDNA, FFPE, blood, cell, or tissue samples

  • Optimized procedure enables high cytosine conversion rates of over 99%
  • Efficient bisulfite conversion in as little as 30 minutes
  • Dedicated solutions for gDNA, whole blood, cells, tissue, and FFPE slices
  • Fast, streamlined procedure for efficient conversion and purification of DNA
  • Unique DNA Protect technology prevents DNA degradation

EpiTect Fast Bisulfite Conversion Kits are a coordinated system of kits that enable highly efficient conversion of unmethylated cytosines to uracils in as little as 30 minutes, while offering outstanding flexibility in choice of starting material. Streamlined procedures for sample lysis and direct bisulfite conversion of DNA from whole blood, cultured cells, tissue samples, genomic DNA, and formalin-fixed, paraffin-embedded (FFPE) slices make efficient bisulfite conversion fast and effortless. Innovative DNA Protect technology ensures effective DNA denaturation without degradation, resulting in high DNA yield and reproducible results. In addition, the converted DNA can be eluted in as little as 10 µl elution buffer, resulting in high DNA concentration. Procedures can be automated on the QIAcube Connect.

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EpiTect Fast Bisulfite Kit (10)
Trial kit for 10 preps: Deparaffinization Solution, Lysis Buffer, Proteinase K, Bisulfite Solution, DNA Protect Buffer, MinElute DNA Spin Columns, Carrier RNA, and Buffers
59802
¥16,000
EpiTect Fast DNA Bisulfite Kit (50)
For 50 preps: Bisulfite Solution, DNA Protect Buffer, MinElute DNA Spin Columns, Carrier RNA, and Buffers
59824
¥40,000
EpiTect Fast DNA Bisulfite Kit (200)
For 200 preps: Bisulfite Solution, DNA Protect Buffer, MinElute DNA Spin Columns, Carrier RNA, and Buffers
59826
¥124,000
EpiTect Fast LyseAll Bisulfite Kit (50)
For 50 preps: Lysis Buffer, Proteinase K, Bisulfite Solution, DNA Protect Buffer, MinElute DNA Spin Columns, Carrier RNA, and Buffers
59864
¥42,000
EpiTect Fast LyseAll Bisulfite Kit (200)
For 200 preps: Lysis Buffer, Proteinase K, Bisulfite Solution, DNA Protect Buffer, MinElute DNA Spin Columns, Carrier RNA, and Buffers
59866
¥142,000
EpiTect Fast FFPE Bisulfite Kit (50)
For 50 preps: Deparaffinization Solution, Lysis Buffer, Proteinase K, Bisulfite Solution, DNA Protect Buffer, MinElute DNA Spin Columns, Carrier RNA, and Buffers
59844
¥65,000

EpiTect Fast Bisulfite Conversion Kits は分子生物学実験用です。疾病の診断、治療または予防の目的には使用することはできません。


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X染色体DNAの高効率シトシン変換|EpiTect Fast DNA Bisulfite Kit操作手順|EpiTect Fast FFPE Bisulfite Kit操作手順|EpiTect Fast LyseAll Bisulfite Kit操作手順|Bisulfite変換後のDNAフラグメントサイズの分析|卓越したBisulfite変換|DNA Protect Buffer|EpiTect Fast Bisufite Conversion Kit|EpiTect Fast Bisulfite Kitの相互関係|
EpiTect Fast DNA Bisulfite Kitを用いてK562ゲノムDNAのBisulfite変換を行ない、X染色体から100 bpの増幅産物をシトシンフリーのプライマーで増幅し、未変換およびBisulfite変換DNAの増幅を行なった。PCR産物をベクターにクローニングし、Sangerシークエンシング法を用いて変換したシトシンを同定した。1252個の個々のシトシンを解析し、シトシン変換効率が99.8%であった。この高い変換効率は、このゲノムDNAが女性由来であるため特に意義がある;すなわち2つのX染色体の一つが高度にメチル化されている。|EpiTect Fast DNA Bisulfite KitはゲノムDNAの完全なBisulfite変換とクリーンアップを効率的かつ簡単に約1時間で実現|脱パラフィン、溶解、クロスリンク切断ステップの完了後、EpiTect Fast DNA Bisulfite Kitを用いてBisulfite変換|サンプル溶解ステップの完了後、添付のEpiTect Fast DNA Bisulfite KitでBisulfite変換を実施|EpiTect Fast DNA Bisulfite Kitを用いたBisulfite変換後、1 µgのゲノムDNAの断片化レベルは最も低く、フラグメントサイズの分布は数百bpから6 kbを超える範囲であったが、他社のキットでは500 bpと1 kbにそれぞれピークがあった。サンプルはAgilent RNA 6000 Nano Chipで解析した。|EpiTect Fast DNA Bisulfite KitあるいはZ社のキットを用いてゲノムDNAのBisulfite変換後、未変換ゲノムDNAとBisulfite変換DNAを一緒に増幅し、その後パイロシークエンシングにより個々のシトシンの変換率を定量した。[A] 2 µgのDNAを解析した場合、EpiTect Fast DNA Bisulfite KitはZ社のキットと同等の結果であった。[B] しかし、解析されたDNAが微量である場合(10 ng)、このパイログラムに示されている4つのシトシン残基は、Z社のキットよりもEpiTect Fast DNA Bisulfite Kitが卓越した結果であることを証明している(Bisulfite変換されなかった各シトシン残基の割合をZ社のキット対EpiTect Fast DNA Bisulfite Kitで比較:それぞれ12%対2%、5%対1%、37%対9%、11%対2%)。|DNA Protect BufferをBisulfite変換反応液に添加すると、バッファーの色が緑色から青色に変わることで溶液が十分に混和され、適切なpH値であることが示される。||サンプルタイプに応じてDNAのBisulfite変換は特化されたキットとプロトコールが必要。キットにはDNA抽出に至適化済みのバッファーとDNAのBisulfite変換に必要な全試薬が含まれている。|
パフォーマンス
Highly efficient bisulfite conversion

Bisulfite treatment of DNA and the resulting conversion of unmethylated cytosines to uracils is the most critical step in methylation analysis, since the reaction efficiency will greatly impact the reliability of downstream analyses. EpiTect Fast Bisulfite Conversion Kits contain innovative Bisulfite Solution, which enables ultrafast, highly efficient bisulfite conversion, even on difficult-to-analyze samples such as X-chromosome genomic DNA derived from a female sample, where one of the 2 X-chromosomes is highly methylated (see figure "Highly efficient cytosine conversion of X chromosome DNA"). Quantification of individual cytosine conversion by Pyrosequencing shows that significantly better conversion is achieved with the EpiTect Fast Bisulfite Conversion Kit than a kit from another supplier, particularly when the amount of starting material is limited (see figure "Superior bisulfite conversion").

Reduced DNA fragmentation

The harsh conditions necessary for complete bisulfite conversion usually lead to a high degree of DNA fragmentation and sample loss, leading to low DNA yield, highly fragmented DNA, and irreproducible conversion rates. QIAGEN's unique DNA Protect Buffer prevents DNA degradation, resulting in fully-converted DNA of much larger fragment sizes than DNA converted with other suppliers' kits (see figure "Analysis of DNA fragment sizes after bisulfite conversion").

原理

Fast and complete bisulfite conversion of sample DNA is essential for accurate DNA methylation analysis. EpiTect Fast Bisulfite Conversion Kits include an innovative Bisulfite Solution that allows highly efficient bisulfite conversion in as little as 30 minutes. Streamlined sample lysis and conversion protocols allow efficient purification and bisulfite conversion of DNA, while unique DNA Protect technology minimizes DNA fragmentation during bisulfite treatment. The 3 EpiTect Fast Bisulfite Conversion Kits are interconnected, functioning as an integrated system of solutions for direct bisulfite conversion of DNA from a broad range of starting materials (see figure "Interconnected protocols of the EpiTect Fast Bisulfite Conversion Kits"). However, each kit has unique features, addresses a specific sample type, and includes all buffers and reagents necessary to function independently. The components in each kit combine to deliver effective lysis of samples, minimal loss of DNA during lysis, complete bisulfite conversion, robust protection during conversion, and thorough cleanup of DNA.

Fast bisulfite conversion

EpiTect Fast Bisulfite Conversion Kits provide a very fast and streamlined procedure for efficient conversion and purification of DNA prepared from genomic DNA, FFPE, blood, cells, or tissue samples. The kits contain highly concentrated Bisulfite Solution, which reduces the time required to convert unmethylated cytosine residues into uracil from several hours to as little as 30 minutes, as well as preparation buffers that make it unnecessary to isolate the DNA prior to bisulfite treatment. Furthermore, the bisulfite thermal cycling program provides an optimized series of incubation steps necessary for thermal DNA denaturation and subsequent sulfonation and cytosine deamination, enabling high cytosine conversion rates of over 99%. Desulfonation, the final step in chemical conversion of cytosines, is achieved by a convenient on-column step included in the purification procedure.

DNA Protect Buffer

Fast and complete bisulfite conversion of sample DNA is the most critical step for the correct determination of a methylation pattern. This is achieved by incubating the DNA in high bisulfite salt concentrations at high temperature and low pH. These harsh conditions often lead to a high degree of DNA fragmentation and subsequent loss of DNA during purification. Common bisulfite procedures usually require high amounts of input DNA to compensate for DNA degradation during conversion and DNA loss during purification, which often lead to low DNA yield, highly fragmented DNA, and irreproducible conversion rates. With EpiTect Fast Bisulfite Conversion Kits, DNA fragmentation is prevented during bisulfite conversion by the unique DNA Protect Buffer, which is uniquely formulated to prevent the fragmentation usually associated with bisulfite treatment of DNA at high temperatures and low pH values. It also provides effective DNA denaturation, resulting in the single-stranded DNA necessary for complete cytosine conversion. DNA Protect Buffer also contains a convenient pH indicator dye as a mixing control allowing confirmation of the correct pH for cytosine conversion (see figure "DNA Protect Buffer").

Deparaffinization of FFPE samples

The first step in extracting DNA from FFPE samples is deparaffinization, whereby paraffin is first dissolved and then removed, exposing samples for subsequent treatment with lysis buffer and proteinase K. With the EpiTect Fast FFPE Bisulfite Kit, the deparaffinization, lysis, and decrosslinking of FFPE slices is optimized with Deparaffinization Solution, an innovative chemistry that facilitates deparaffinization with minimal handling. Deparaffinization Solution contributes to high DNA recovery rates, since it remains on the sample while proteinase K digestion is carried out. With no need to pellet the FFPE sample or to remove the paraffin-containing supernatant, the risk of sample loss during deparaffinization is avoided.

操作手順

EpiTect Fast Bisulfite Conversion Kit procedures comprise a few simple steps: preparation of DNA from a sample, bisulfite-mediated conversion of unmethylated cytosines; binding of the converted single-stranded DNA to the membrane of a MinElute DNA spin column; washing; desulfonation of membrane-bound DNA; washing of the membrane-bound DNA to remove the desulfonation agent; and elution of the pure, converted DNA from the spin column. Sample preparation is different for FFPE slices, whole blood, cell cultures, or tissues, whereas the procedure for bisulfite conversion of extracted DNA is the same for all sample types (see figure "Interconnected protocols of the EpiTect Fast DNA Conversion Kits"). All protocols achieve the same cytosine conversion rates and lead to equal DNA recoveries after purification of converted DNA, independent of DNA starting amounts.

Kit description

EpiTect Fast DNA Bisulfite Kit

The EpiTect Fast DNA Bisulfite Kit includes carefully formulated buffers and reagents to provide streamlined and effective bisulfite conversion of genomic DNA and subsequent cleanup of the converted DNA (see Figure "EpiTect Fast DNA Bisulfite Kit procedure"). The novel Bisulfite Solution enables complete bisulfite conversion in as little as 30 minutes, while DNA Protect technology and QIAGEN spin columns allow the generation of fully converted DNA with minimal degradation that can be eluted in as little as 10 µl of buffer for downstream applications requiring high DNA concentrations. The EpiTect Fast DNA Bisulfite Kit can be used for conversion of 1 ng–2 µg DNA in a volume of up to 20 µl or 1–500 ng in a maximum volume of 40 µl.

EpiTect Fast FFPE Bisulfite Kit

Formalin-fixed, paraffin-embedded (FFPE) tissues are processed with the EpiTect Fast FFPE Bisulfite Kit, which consists of the EpiTect Fast FFPE Lysis Kit, containing specialized buffers for efficient deparaffinization and lysis of FFPE tissue slices (see figure "EpiTect Fast FFPE Bisulfite Kit procedure"), and the EpiTect Fast DNA Bisulfite Kit for fast and effective bisulfite conversion of the extracted DNA. The protocol includes an optimized step to facilitate binding of DNA and can be used with single slices of FFPE tissue (10 µm in thickness). The DNA fragmentation that inevitably occurs when DNA is extracted from FFPE tissues is made worse by the harsh conditions of subsequent bisulfite treatment. QIAGEN's reliable DNA Protect technology minimizes fragmentation during the conversion reaction, resulting in superior yields of bisulfite converted DNA.

EpiTect Fast LyseAll Bisulfite Kit

Whole blood, cultured cells, or tissue samples are processed with the EpiTect Fast LyseAll Bisulfite Kit, which consists of the EpiTect Fast LyseAll Lysis Kit (see figure "EpiTect Fast LyseAll Bisulfite Kit procedure") with an innovative lysis buffer and the EpiTect Fast DNA Bisulfite Kit for bisulfite conversion of the extracted DNA. The protocol includes an optimized step to facilitate binding of DNA along with uniquely formulated lysis buffers to streamline the release of DNA from samples. This kit can be used with 0.5–20 µl blood or 10–105 cells (as little as 60 pg of DNA).

アプリケーション
The EpiTect Fast Bisulfite Conversion Kit delivers fast and efficient bisulfite conversion of DNA from various starting material that is highly suited for all techniques currently used for the analysis of DNA methylation, including:
  • PCR: end-point, real-time, and methylation-specific
  • Bisulfite sequencing (direct and cloning)
  • COBRA (Combined Bisulfite Restriction Analysis)
  • Pyrosequencing

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キットハンドブック
1
For sample lysis and complete bisulfite conversion/cleanup of DNA from FFPE, blood, cultured cells, or tissue samples, optimized for methylation analysis
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パンフレット
1
High-quality, nucleic acid purification for successful PCR and NGS experiments.
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Highly efficient cytosine conversion of  X-chromosome DNA
X染色体DNAの高効率シトシン変換
EpiTect Fast DNA Bisulfite Kitを用いてK562ゲノムDNAのBisulfite変換を行ない、X染色体から100 bpの増幅産物をシトシンフリーのプライマーで増幅し、未変換およびBisulfite変換DNAの増幅を行なった。PCR産物をベクターにクローニングし、Sangerシークエンシング法を用いて変換したシトシンを同定した。1252個の個々のシトシンを解析し、シトシン変換効率が99.8%であった。この高い変換効率は、このゲノムDNAが女性由来であるため特に意義がある;すなわち2つのX染色体の一つが高度にメチル化されている。
EpiTect Fast DNA Bisulfite Kit procedure
EpiTect Fast DNA Bisulfite Kit操作手順
EpiTect Fast DNA Bisulfite KitはゲノムDNAの完全なBisulfite変換とクリーンアップを効率的かつ簡単に約1時間で実現
EpiTect Fast FFPE Bisulfite Kit procedure
EpiTect Fast FFPE Bisulfite Kit操作手順
脱パラフィン、溶解、クロスリンク切断ステップの完了後、EpiTect Fast DNA Bisulfite Kitを用いてBisulfite変換
EpiTect Fast  LyseAll Bisulfite Kit procedure
EpiTect Fast LyseAll Bisulfite Kit操作手順
サンプル溶解ステップの完了後、添付のEpiTect Fast DNA Bisulfite KitでBisulfite変換を実施
Analysis of DNA fragment sizes after bisulfite conversion
Bisulfite変換後のDNAフラグメントサイズの分析
EpiTect Fast DNA Bisulfite Kitを用いたBisulfite変換後、1 µgのゲノムDNAの断片化レベルは最も低く、フラグメントサイズの分布は数百bpから6 kbを超える範囲であったが、他社のキットでは500 bpと1 kbにそれぞれピークがあった。サンプルはAgilent RNA 6000 Nano Chipで解析した。
Superior bisulfite conversion
卓越したBisulfite変換
EpiTect Fast DNA Bisulfite KitあるいはZ社のキットを用いてゲノムDNAのBisulfite変換後、未変換ゲノムDNAとBisulfite変換DNAを一緒に増幅し、その後パイロシークエンシングにより個々のシトシンの変換率を定量した。[A] 2 µgのDNAを解析した場合、EpiTect Fast DNA Bisulfite KitはZ社のキットと同等の結果であった。[B] しかし、解析されたDNAが微量である場合(10 ng)、このパイログラムに示されている4つのシトシン残基は、Z社のキットよりもEpiTect Fast DNA Bisulfite Kitが卓越した結果であることを証明している(Bisulfite変換されなかった各シトシン残基の割合をZ社のキット対EpiTect Fast DNA Bisulfite Kitで比較:それぞれ12%対2%、5%対1%、37%対9%、11%対2%)。
DNA Protect Buffer
DNA Protect BufferをBisulfite変換反応液に添加すると、バッファーの色が緑色から青色に変わることで溶液が十分に混和され、適切なpH値であることが示される。
EpiTect Fast Bisufite Conversion Kit
Interconnected protocols of the EpiTect Fast Bisulfite Conversion Kits
EpiTect Fast Bisulfite Kitの相互関係
サンプルタイプに応じてDNAのBisulfite変換は特化されたキットとプロトコールが必要。キットにはDNA抽出に至適化済みのバッファーとDNAのBisulfite変換に必要な全試薬が含まれている。