AllPrep DNA/RNA FFPE Kit

For simultaneous purification of genomic DNA and total RNA (including small RNAs) from formalin-fixed, paraffin-embedded tissue sections
  • 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
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.
製品名 Cat. no. List price:
AllPrep DNA/RNA FFPE Kit (50)
50 RNeasy MinElute Spin Columns, 50 QIAamp MinElute Spin Columns, Collection Tubes, RNase-Free Reagents, and Buffers
80234
¥75,500

AllPrep DNA/RNA FFPE Kit  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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Purification of DNA and RNA from FFPE samples.|Array analysis following preamplification of cDNA targets.|Reliable amplification of DNA and RNA from FFPE samples.|AllPrep DNARNA FFPE procedure.|
[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.|Total RNA was purified from human breast FFPE tissue using the AllPrep DNA/RNA FFPE Kit. RNA was then reverse-transcribed using RT2 FFPE PreAMP technology. Gene expression analysis by real-time PCR was performed using the Human Cell Cycle RT2 Profiler PCR Array, comparing a tumor sample to a nontumor sample. ΔΔCT analysis shows the x-fold difference in gene expression of tumor sample compared to nontumor sample.|[A] DNA and [B], [C] RNA were purified from various FFPE rat tissues using either the AllPrep DNA/RNA FFPE Kit or, as a control, dedicated kits for DNA or RNA purification from FFPE samples (QIAamp DNA FFPE Tissue Kit or RNeasy FFPE Kit). Real-time PCR or real-time RT-PCR was carried out on an ABI PRISM 7900HT Sequence Detection System using [A] the QuantiTect SYBR® Green PCR Kit to analyze a 78 bp amplicon of the Prnp gene or [B], [C] the QuantiTect SYBR® Green RT-PCR Kit to analyze Jun oncogene expression. The AllPrep Kit and the dedicated kits provided comparable CT values, indicating that all kits achieved similar efficiency in recovering usable DNA or RNA. [C] In addition, analysis of Jun expression was carried out without reverse transcriptase (-RT). The amplification plot for the spleen sample, a DNA-rich tissue, indicated the virtual absence of genomic DNA contamination.||
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 "Purification of DNA and RNA from FFPE samples"). The purified nucleic acids are therefore suitable for downstream applications such as Pyrosequencing or real-time PCR and RT-PCR (see figure "Reliable amplification of DNA and RNA from FFPE samples" and "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 "AllPrep DNA/RNA FFPE procedure"). 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.
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.

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FFPE サンプルの分子解析における重要なファクター
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Sample to Insight solutions for successful molecular analysis
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Critical factors for molecular analysis of FFPE samples
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High-quality, nucleic acid purification for successful PCR and NGS experiments.
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キットハンドブック
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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
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Purification of DNA and RNA from FFPE samples
FFPE サンプルからDNA およびRNA の精製
[A] 室温で表記期間保存した様々なラットFFPE 組織からゲノムDNA を精製した。AllPrep DNA/RNA FFPE KitあるいはコントロールとしてQIAamp DNA FFPE Tissue Kit(FFPEサンプルからのDNA精製専用キット)を用いて精製し、両キットともにRNase分解ステップを実施した。各サンプルの切片(20 μm)から得たDNA 収量を、A260 の吸光度を測定して計算した。[B] 室温で表記期間保存した様々なラットFFPE 組織からRNA を精製した。AllPrep DNA/RNA FFPE KitあるいはコントロールとしてRNeasy FFPE Kit(FFPEサンプルからのRNA精製専用キット)を用いて精製した。各サンプルの切片(10 μm)から得たRNA 収量を、A260 の吸光度を測定して計算した。AllPrep Kitは、FFPEサンプルからDNA/RNAをそれぞれ分離精製する専用のDNA/RNA精製キットと同様の性能を示した。
Array analysis following preamplification of cDNA targets
cDNAターゲットの事前増幅後のアレイ解析
AllPrep DNA/RNA FFPE Kitを用いてヒト乳房FFPE組織からトータルRNAを精製した。RT2 FFPE PreAMPテクノロジーを使用してRNAを逆転写し、事前増幅を行なった。リアルタイムPCRによる遺伝子発現解析は、Human Cell Cycle RT2  Profiler PCR Arrayを用いてリアルタイムPCRによる遺伝子発現を行ない、健常組織とがん組織を比較した。ΔΔCT 解析の結果、健常組織に比較してがん組織の遺伝子発現に数倍の差異が示された。
Reliable amplification of DNA and RNA from FFPE samples
FFPE サンプルからDNA およびRNA の信頼性のある増幅
AllPrep DNA/RNA FFPE Kitと、コントロールとしてFFPEサンプルからのDNAまたはRNA精製専用キット(QIAamp DNA FFPE Tissue KitあるいはRNeasy FFPE Kit)を用いて様々なラットFFPE組織からDNA[A]およびRNA [B]、[C]を精製した。Prnp 遺伝子の増幅産物(78 bp)の解析にはQuantiTect SYBR® Green PCR Kit [A]を、Jun癌遺伝子の発現解析にはQuantiTect SYBR® Green RT-PCR Kit [B]、 [C]を用いてABI PRISM 7900HT Sequence Detection SystemでリアルタイムPCR/RT-PCRを行った。AllPrep Kitによる精製は、専用キットで精製した核酸で得られたCT値と同等であったことから、同等品質の有効なDNAやRNAを効率よく精製できることを示唆している。[C]さらに逆転写酵素なし(-RT)でJun発現解析を行なった。DNAリッチな組織である脾臓サンプルの増幅曲線はゲノムDNAコンタミが実質上ないことを示した。
AllPrep DNA/RNA FFPE procedure
AllPrep DNA/RNA FFPE操作手順