QIAsymphony Bisulfite Kits

• Fast and streamlined combined bisulfite conversion and DNA purification
• Directly isolate purified, bisulfite-converted DNA without intermediary steps
• Produces high-quality, bisulfite-converted DNA suitable for pyrosequencing, PCR or NGS applications
• High cytosine conversion rates of over 99%

QIAsymphony Bisulfite Kits provide a fast, streamlined procedure for efficient bisulfite conversion and purification of DNA prepared from formalin-fixed, paraffin-embedded (FFPE), blood, cell, or tissue samples. The kits enable highly efficient conversion of unmethylated cytosines to uracils in as little as 30 minutes while offering outstanding flexibility in choice of starting material. Using the QIAsymphony SP instrument, isolate high-quality genomic DNA that is suitable for direct use in downstream applications, such as Pyrosequencing, PCR or NGS applications, or for storage for later use.

Highly efficient bisulfite conversion

The methylation status of a DNA sequence can best be determined using bisulfite conversion. Bisulfite treatment of the target DNA results in conversion of unmethylated cytosine residues into uracil, leaving the methylated cytosines unchanged. Therefore, bisulfite treatment gives rise to different DNA sequences for methylated and unmethylated DNA. Bisulfite treatment of DNA is the most critical step in methylation analysis, since the reaction efficiency will greatly impact the reliability of downstream analyses. QIAsymphony Bisulfite Kits contain innovative Bisulfite Solution, which enables ultrafast, highly efficient 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.

Broad sample acceptance and application spectrum

While distinct protocols are used for FFPE slices, whole blood and cell cultures or tissues, these protocols achieve the same cytosine conversion rates and lead to equal DNA recoveries after purification of converted DNA, independent of DNA starting amounts. The purified DNA is ready to use in downstream applications. The eluted, bisulfite-converted DNA is suited for all techniques currently used for the analysis of DNA methylation, including PCR, real-time PCR, methylation-specific PCR, bisulfite sequencing (direct and cloning), COBRA, Pyrosequencing and NGS applications.

Fast and complete bisulfite conversion of sample DNA is essential for accurate DNA methylation analysis. QIAsymphony Bisulfite 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 QIAsymphony Bisulfite Kits are interconnected, functioning as an integrated system of solutions for direct bisulfite conversion of DNA from a broad range of starting materials.

Fast bisulfite conversion

QIAsymphony Bisulfite 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-beads step included in the purification procedure on the QIAsymphony SP.

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 QIAsymphony Bisulfite 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.

QIAsymphony procedure

The QIAsymphony Bisulfite Kit procedure simplifies sample preparation and isolation following bisulfite conversion using the QIAsymphony SP instrument (see figure “Schematic of the QIAsymphony SP principle”). The QIAsymphony SP can save you time by processing 1–96 samples — in batches of 24. QIAsymphony technology combines the speed and efficiency of silica-based purification with the convenient handling of magnetic particles.

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 QIAsymphony Bisulfite FFPE 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.

The QIAsymphony Bisulfite Kit procedure simplifies sample preparation and isolation following bisulfite conversion using the QIAsymphony SP instrument (see figure “Schematic of the QIAsymphony SP principle”). It comprises a few simple steps: preparation of DNA from sample, bisulfite-mediated conversion of unmethylated cytosines (see figure “Bisulfite reaction set up”), transfer of sample to the QIAsymphony SP module, automated binding of the converted single-stranded DNA to the magnetic beads, washing, desulfonation of beads-bound DNA, washing of the bead-bound DNA to remove the desulfonation agent and elution of the pure, converted DNA from the beads (see figure “Automated bisulfite conversion on a QIAsymphony SP”).

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. All protocols achieve the same cytosine conversion rates and lead to equal DNA recoveries after purification of converted DNA, independent of DNA starting amounts.

QIAsymphony Bisulfite Kit

The QIAsymphony 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. The novel Bisulfite Solution enables complete bisulfite conversion in as little as 30 minutes, while DNA Protect technology allow the generation of fully converted DNA with minimal degradation. The QIAsymphony 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.

QIAsymphony Bisulfite FFPE Kit

Formalin-fixed, paraffin-embedded (FFPE) tissues are processed with the QIAsymphony Bisulfite FFPE Kit, which consists of the EpiTect Fast FFPE Lysis Kit, containing specialized buffers for efficient deparaffinization and lysis of FFPE tissue slices, and the QIAsymphony 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.

QIAsymphony Bisulfite LyseAll Kit

Whole blood, cultured cells, or tissue samples are processed with the QIAsymphony Bisulfite LyseAll Kit, which consists of the EpiTect Fast LyseAll Lysis Kit with an innovative lysis buffer and the QIAsymphony 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 QIAsymphony Bisulfite Kits deliver 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)
• NGS
• Pyrosequencing
• COBRA (Combined Bisulfite Restriction Analysis)