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AllPrep DNA/RNA FFPE Kit is intended for simultaneous purification of genomic DNA and total RNA (including small RNAs) from formalin-fixed, paraffin-embedded tissue sections in molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
AllPrep DNA/RNA FFPE Kit uses a patent-pending solubilization method to purify DNA and RNA from formalin-fixed, paraffin-embedded (FFPE) tissue sections. Purified analytes are suitable for use in applications such as real-time PCR and Pyrosequencing. For reliable comparison of genomic and transcriptomic data, purification of DNA and RNA from the same sample is essential. The kit can be automated on the QIAcube Connect MDx.
DNA and RNA purified using the AllPrep DNA/RNA FFPE Kit are of comparable quality to DNA and RNA purified using the QIAamp FFPE Tissue Kit and RNeasy FFPE Kit/miRNeasy FFPE, respectively. The purified nucleic acids are therefore suitable for downstream applications such as Pyrosequencing or real-time PCR and RT-PCR
A simple workflow allows the purification of high-quality DNA and RNA from the same FFPE tissue section sample. The AllPrep DNA/RNA FFPE Kit uses a patent-pending solubilization method to differentially release DNA and RNA from a single FFPE sample. With this method, FFPE samples are incubated in an optimized lysis buffer, which results in the release of RNA and precipitation of DNA. After centrifugation, the RNA-containing supernatant and DNA-containing pellet are then processed separately to purify RNA and DNA. Further incubations partially reverse crosslinking, and RNA or DNA is then purified using an RNeasy MinElute spin column or QIAamp MinElute spin column. For purified RNA, an on-column DNase treatment efficiently removes any contaminating DNA. Depending on the RNA binding conditions, small RNAs such as miRNA are either absent or present in the purified RNA. For purified DNA, an on-column RNase treatment is optional, as RNA contamination is minimal due to the separation of DNA and RNA prior to spin column processing.
The AllPrep DNA/RNA FFPE Kit is specially designed for simultaneous purification of genomic DNA and total RNA from FFPE tissue sections. Pure DNA and RNA are obtained from the entire sample, in contrast to other procedures where the biological sample is divided into two before being processed separately. Simply dividing a sample in half for separate DNA and RNA purification procedures results in the purification of DNA and RNA from different populations of cells, which may differ in their properties. Purification of DNA and RNA from the same sample also helps to prevent waste, since FFPE samples are precious, often difficult to retrieve, and limited in amount.
Due to fixation and embedding conditions, nucleic acids in FFPE samples are usually heavily fragmented and are often of a lower molecular weight than those obtained from fresh or frozen samples. A major obstacle to isolating DNA and RNA from the same FFPE sample is that fragmented DNA is short and can be partly single-stranded and therefore more closely resembles RNA than intact DNA. This property of fragmented DNA makes physical separation of DNA and RNA difficult. The AllPrep DNA/RNA FFPE Kit uses a patent-pending solubilization method to differentially release DNA and RNA from a single FFPE sample.
Nucleic acids in FFPE samples are also chemically modified by formaldehyde. Although formaldehyde modification cannot be detected in standard quality control assays, such as gel electrophoresis or lab-on-a-chip analysis, it does strongly interfere with enzymatic analyses. The AllPrep DNA/RNA FFPE Kit is optimized to reverse as much formaldehyde modification as possible without further DNA and RNA degradation.
|Applications||PCR, qPCR, real-time RT-PCR, microarray|
|Main sample type||FFPE tissue samples|
|Elution volume||RNA: 14-30µl ; DNA: 30-100µl|
|Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein||RNA (miRNA) and DNA|
|Number of preps per run||50|
|Sample amount||max. 4*10 µm sections or 2*20 µm sections|
|Time per run or per prep||6h for 10 samples, including sectioning|