AllPrep DNA/RNA FFPE Kit – Nucleic Acid Extraction

For simultaneous purification of genomic DNA and total RNA (including small RNAs) from formalin-fixed, paraffin-embedded tissue sections

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AllPrep DNA/RNA FFPE Kit (50)

Cat. No. / ID: 80234

50 RNeasy MinElute Spin Columns, 50 QIAamp MinElute Spin Columns, Collection Tubes, RNase-Free Reagents, and Buffers

The AllPrep DNA/RNA FFPE Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Want to try this solution for the first time?

Get in touch with our team today and request a quote for your AllPrep DNA/RNA FFPE Kit (50) trial kit.

Request a quote for a trial kit

Features

Maximum output with minimal sample consumption
Releases DNA/RNA without compromising integrity
Effectively separates RNA and DNA
Comprehensive DNA and RNA analysis of the same FFPE sample

Product Details

For reliable comparison of genomic and transcriptomic data, purification of DNA and RNA from the same sample is essential. The 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. The kit can be automated on the QIAcube Connect.

Performance

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 (see figure ""). The purified nucleic acids are therefore suitable for downstream applications such as Pyrosequencing or real-time PCR and RT-PCR (see figure "" and "").

See figures

Purification of DNA and RNA from FFPE samples.

Reliable amplification of DNA and RNA from FFPE samples.

Array analysis following preamplification of cDNA targets.

Principle

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.

Procedure

A simple workflow allows the purification of high-quality DNA and RNA from the same FFPE tissue section sample (see flowchart ""). 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.

See figures

AllPrep DNARNA FFPE procedure.

Applications

The AllPrep DNA/RNA FFPE Kit is optimized to reverse as much formaldehyde modification as possible without further DNA and RNA degradation; however nucleic acids purified from FFPE samples should not be used in downstream applications that require high-molecular-weight DNA or full-length RNA. Some applications may require modifications to allow the use of fragmented nucleic acids (e.g., designing small amplicons for PCR and RT-PCR). For cDNA synthesis, gene-specific primers should be used instead of oligo-dT primers. If it is not possible to use gene-specific primers, random primers should be used.

Supporting data and figures

Purification of DNA and RNA from FFPE samples.

[A] Genomic DNA was purified from various FFPE rat tissues that were stored at room temperature for the times indicated. Purification was performed using either the AllPrep DNA/RNA FFPE Kit or, as a control, the QIAamp DNA FFPE Tissue Kit (a dedicated kit for DNA purification from FFPE samples) both including RNase digestion. DNA yields from 20 μm sections of each sample were determined by absorbance measurement. [B] RNA was purified from various FFPE rat tissues that were stored at room temperature for the times indicated. Purification was performed using either the AllPrep DNA/RNA FFPE Kit or, as a control, the RNeasy FFPE Kit (a dedicated kit for RNA purification from FFPE samples). RNA yields from 10 μm sections of each sample were determined by absorbance measurement. The AllPrep Kit performed just as well as the dedicated DNA/RNA purification kits in recovering DNA/RNA from FFPE samples.

Specifications

Features	Specifications
Applications	PCR, qPCR, real-time RT-PCR, microarray
Elution volume	RNA: 14-30µl ; DNA: 30-100µl
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein	RNA (miRNA) and DNA
Processing	Manual (centrifugation)
Format	Spin column
Number of preps per run	50
Sample amount	max. 410 µm sections or 220 µm sections
Technology	Silica technology
Time per run or per prep	6h for 10 samples, including sectioning
Yield	Varies
Main sample type	FFPE tissue samples

Resources

Sample to Insight solutions for successful molecular analysis

High-quality, nucleic acid purification for successful PCR and NGS experiments.

FFPE サンプルの分子解析における重要なファクター

High-quality, nucleic acid purification for successful PCR and NGS experiments.

Critical factors for molecular analysis of FFPE samples

ホルマリン固定パラフィン包埋（FFPE）組織切片からのゲノムDNA およびトータルRNA（small RNA を含む）の同時精製

FAQ

The most important components of Buffer FRN are guanidine thiocyanate and isopropanol. Isopropanol is added by the user before using the AllPrep DNA/RNA FFPE Kit for the first time. The exact composition of Buffer FRN is confidential.

FAQ ID -2802

The exact composition of Buffer PKD is proprietary. Buffer PKD functions as a Proteinase K Digestion Buffer and is a component of, for example, the AllPrep DNA/RNA FFPE Kit, RNeasy FFPE Kit, and the miRNeasy FFPE Kit. We are sometimes asked if Buffer PKD comprises any RNase inhibitors or RNase inhibiting agents — since the formalin-fixation of the starting material has already inactivated the RNases, no such reagents are present in this buffer.

FAQ ID -2801

DNA and RNA yield from FFPE tissue depends highly on the tissue type, fixation, embedding, storage conditions, and the age of the sample. Thus, no general values for DNA or RNA yield can be given.

FAQ ID -2353

Purified DNA can be stored at 2 to 8°C for up to 24 hours before use in downstream applications and should be placed at –20°C for longer storage. Purified RNA should be stored at –20oC or –70oC in RNase-free water.

FAQ ID -2350

Yes, gained yields and quality are comparable. Separation of RNA from DNA in the AllPrep protocol is highly effective. As A260 values measure both DNA and RNA, lower A260 values from DNA purified using the AllPrep DNA/RNA FFPE Kit may indicate high DNA purity and the absence of contaminating RNA. Higher A260 values from DNA purified using alternative methods may indicate the presence of significant amounts of contaminating RNA.

FAQ ID -2349

Prior to nucleic acid purification, the paraffin in an FFPE sample needs to be removed to allow exposure of the sample to proteinase K. Deparaffinization can either be performed using QIAGEN’s Deparaffinization Solution (recommended), heptane-methanol, or xylene.

FAQ ID -2352

Yes. The AllPrep DNA/RNA FFPE Kit includes a special protocol for micro RNA purification.

FAQ ID -2348

With the AllPrep DNA/RNA FFPE Kit up to 4*10µm sections or 2*20µm sections with a maximum surface area of 150mm² can be used. Thicker or larger sections may result in lower nucleic acid yields, even after prolonged incubation with proteinase K.

FAQ ID -2351

Tissue-Tek O.C.T. is an embedding compound for cryosectioning, which is soluble in water. It mainly consists of glycols and synthetic resins. Tissue-Tek O.C.T. is used as matrix for cryosectioning of tissues. Using the O.C.T. the tissue samples can be positioned more easily in the microtome and have better qualities during sectioning.

Most cryosections are fixed using non-crosslinking agents. For isolation of RNA from tissue embedded in Tissue-Tek O.C.T. using non-crosslinking agents the RNeasy Plus Micro Kit or the RNeasy Micro Kit (Protocol: Total RNA Isolation from Microdissected Cryosections), or the RNeasy Mini Kit (RNeasy Mini Protocol for the isolation of Total RNA from Animal Tissue) give great results. We strongly recommend removing as much of the embedding compound as possible prior to RNA extraction from the sections. Please see QIAGEN News article, Issue 1 1998, 'Effects of malnutrition on expression of lactase in children' for successful RNA isolation from O.C.T.-embedded tissue using the RNeasy Mini Kit.

In case crosslinking agents (e.g. formaldehyde or glyoxal-containing) were used for fixation of the tissue for cryosectioning the RNeasy FFPE Kit is the perfect choice. The RNeasy FFPE Kit is especially designed for purifying total RNA from formalin-fixed tissue sections. Special lysis and incubation conditions reverse formaldehyde modification of RNA for improved results in downstream application. The crosslinking causes the RNA to break, resulting in overall smaller molecules, which look like a smear when analyzed on a formaldehyde gel.

In case DNA as well as RNA from precious samples is of interest from the identical sections, please have a look at the AllPrep DNA/RNA Kits for unfixed tissue or the AllPrep DNA/RNA FFPE Kit for fixed tissue.

FAQ ID -530