EpiTect Fast Bisulfite Conversion Kits

• 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, also in 96-well plates
• 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. The spin-column procedures can be automated on the QIAcube Connect. The procedure in 96-well format delivers ultrafast, high-throughput bisulfite conversion of DNA and can be processed using a centrifuge or vacuum manifold.

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").optimized RT mix for efficient and sensitive reverse transcription over a wide range of RNA template amounts. For convenience, the master mix can be stored at 2–8°C.

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 and EpiTect Fast 96 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 figures  "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.

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 or to the membrane of an EpiTect 96 plate; 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 or 96-well plate. 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 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 descriptions

EpiTect Fast DNA Bisulfite Kit and EpiTect Fast 96 DNA Bisulfite Kit

EpiTect Fast DNA Bisulfite Kits include carefully formulated buffers and reagents to provide streamlined and effective bisulfite conversion of genomic DNA and subsequent cleanup of the converted DNA (see figures  "EpiTect Fast DNA Bisulfite Kit procedure" and  “EpiTect Fast 96 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 or 96-well plates 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. EpiTect Fast DNA Bisulfite Kits 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 (spin-column format only)

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).

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