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.
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AllPrep DNA/RNA FFPE Kit (50)
50 RNeasy MinElute Spin Columns, 50 QIAamp MinElute Spin Columns, Collection Tubes, RNase-Free Reagents, and Buffers
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80234
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AllPrep DNA/RNA FFPE Kit适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。
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.||
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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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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图片
从FFPE样本中纯化DNA和RNA。
[A] 从多个大鼠FFPE组织样本中纯化基因组DNA并储存于室温。使用AllPrep DNA/RNA FFPE Kit或QIAamp DNA FFPE Tissue Kit(专用于从FFPE样本中纯化DNA的试剂盒,作为对照)进行纯化,两种试剂盒都含有RNase消化试剂。采用吸光度测定方法检测每个20 μm组织切片的DNA产量。[B] 从多个大鼠FFPE组织样本中纯化RNA并储存于室温。使用AllPrep DNA/RNA FFPE Kit或RNeasy FFPE Tissue Kit(专用于从FFPE样本中纯化DNA的试剂盒,作为对照)进行纯化。采用吸光度测定方法检测每个10 μm组织切片的RNA产量。从FFPE组织中纯化DNA或RNA时,AllPrep Kit的表现与专用的纯化试剂盒表现相当。
对预扩增的cDNA进行芯片分析。
使用AllPrep DNA/RNA FFPE Kit从人类乳腺FFPE组织中纯化总RNA。然后使用RT2 FFPE PreAMP技术进行逆转录。使用Human Cell Cycle RT2 Profiler PCR Array,通过real-time PCR进行基因表达分析,将肿瘤样本与非肿瘤样本进行对比。ΔΔCT分析显示:肿瘤样本中的基因表达与非肿瘤样本的基因表达存在x倍的差异。
对FFPE样本中的DNA和RNA进行可靠扩增。
使用AllPrep DNA/RNA FFPE Kit或专用于从FFPE样本中纯化DNA或RNA的试剂盒(QIAamp DNA FFPE Tissue Kit 或RNeasy FFPE Kit,作为对照)从大鼠FFPE组织中纯化获得[A] DNA 和[B]、[C] RNA。在ABI PRISM 7900HT Sequence Detection System上,使用[A] QuantiTect SYBR® Green PCR Kit对78 bp的Prnp基因扩增子进行Real-time PCR分析,或是用[B]、[C] QuantiTect SYBR® Green RT-PCR Kit对Jun原癌基因表达进行real-time RT-PCR分析。AllPrep Kit和另外两个专用试剂盒的CT值相当,表明三种试剂盒纯化DNA或RNA的效率相当。[C] 此外,在没有逆转录酶(–RT)的条件下分析了Jun基因的表达。脾脏是富含DNA的组织,其扩增曲线平坦,说明纯化的RNA中不含基因组DNA。
AllPrep DNARNA FFPE样本纯化步骤。
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