从福尔马林固定、石蜡包埋的组织切片中同时纯化基因组DNA和总RNA(包括小RNAs)

  • 使用小样本量即可获得大量纯化产物
  • 在纯化同时,保持DNA和RNA的完整性
  • 高效分离RNA和DNA
  • 对同一FFPE样本进行全面的DNA和RNA分析

如要对基因组学和转录组学数据进行可靠比较,则需从同一样本中纯化DNA和RNA。AllPrep DNA/RNA FFPE Kit使用先进的溶解方法,从福尔马林固定、石蜡包埋的组织样本中纯化DNA和RNA。纯化产物适用于real-time PCR和焦磷酸测序等应用。

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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
80234
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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.
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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
使用AllPrep DNA/RNA FFPE Kit纯化的DNA和RNA的质量与使用QIAamp FFPE Tissue Kit和RNeasy FFPE Kit/miRNeasy FFPE Kit纯化的产物的质量相当(参见"Purification of DNA and RNA from FFPE samples")。因此,纯化产物可用于焦磷酸测序、real-time PCR和RT-PCR等下游应用(参见"Reliable amplification of DNA and RNA from FFPE samples"和"Array analysis following preamplification of cDNA targets")。
Principle

AllPrep DNA/RNA FFPE Kit专为从FFPE样本中同时纯化基因组DNA和总RNA而设计。该试剂盒可使用同一样本纯化DNA和RNA,而非像其他纯化方法那样,需将样本分为两份再分别进行纯化操作。如将样本分为两份分别纯化DNA和RNA,其纯化出的DNA和RNA来自不同的细胞群落,可能具有不同性质特征。从同一样本中同时纯化DNA和RNA可减少浪费,因为FFPE样本是十分珍贵的样本,通常很难恢复且样本量少。

由于固定和包埋,FFPE样本中的核酸通常都严重片段化,核酸的分子量通常小于从新鲜或冷冻样本中分离出的核酸的分子量。从同一FFPE样本中分离DNA和RNA面临着很大困难:片段化的DNA长度短,且部分是单链结构;因此其更像RNA,而非完整的DNA。片段化DNA这一特性使得用物理方法分离DNA和RNA十分困难。AllPrep DNA/RNA FFPE Kit使用先进的溶解方法,从同一个FFPE样本中分别释放DNA和RNA。

尽管标准的质量控制分析(如凝胶电泳或芯片分析方法)无法检测到甲醛的化学修饰,但其对酶分析有严重干扰。AllPrep DNA/RNA FFPE Kit已经过优化,可最大程度上逆转甲醛化学修饰,而不引起DNA和RNA的进一步降解。

Procedure
简单的流程可从同一个样本中纯化高品质DNA和RNA(参见"AllPrep DNA/RNA FFPE procedure")。AllPrep DNA/RNA FFPE Kit使用先进的溶解方法,从同一个FFPE样本中分别释放DNA和RNA。使用这种方法时,FFPE样本要在优化的裂解缓冲液中孵育,使得RNA释放出来,而DNA形成沉淀。离心后,将含有RNA的上清和含有DNA的沉淀分别处理,纯化RNA和DNA。再次进行孵育有一定的解交联作用,然后使用RNeasy MinElute或QIAamp MinElute离心柱纯化RNA和DNA。用柱上DNA酶处理纯化的RNA,以有效去除DNA污染。根据RNA与离心柱的结合情况,纯化的RNA中可能含有也可能不含miRNA等小RNAs。对于纯化后的DNA,可选择性进行柱上RNA酶消化,因为在离心前的DNA和RNA分离步骤中RNA污染水平已达到最低。
Applications

尽管AllPrep DNA/RNA FFPE Kit已经过优化,可最大程度上逆转福尔马林化学修饰,而不引起DNA和RNA的进一步降解;但从FFPE样本中纯化获得的核酸不应该用于需要大分子量DNA或完整长度RNA的下游应用。一些应用可能需要进行化学修饰后,再使用片段化核酸(如:设计PCR和RT-PCR的小扩增子)。在cDNA合成中,应使用基因特异性引物,而非oligo-dT引物。如果无法使用基因特异性引物,则可使用任意引物。

Feature
Specifications
Applications PCR, qPCR, real-time RT-PCR, microarray
Elution volume RNA: 14-30µl ; DNA: 30-100µl
Format Spin column
Main sample type FFPE tissue samples
Number of preps per run 50
Processing Manual (centrifugation)
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein RNA (miRNA) and DNA
Sample amount max. 4*10 µm sections or 2*20 µm sections
Technology Silica technology
Time per run or per prep 6h for 10 samples, including sectioning
Yield Varies

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Critical factors for molecular analysis of FFPE samples
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