AllPrep DNA/RNA FFPE Kit – Nucleic Acid Extraction

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）

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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

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AllPrep DNA/RNA FFPE Kit 旨在用于分子生物学应用。该产品不能用于疾病诊断、预防和治疗。

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特点

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

产品详情

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。

绩效

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""）。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""和""）。

查看图表

Purification of DNA and RNA from FFPE samples.

Reliable amplification of DNA and RNA from FFPE samples.

Array analysis following preamplification of cDNA targets.

原理

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。

程序

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 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。

查看图表

AllPrep DNARNA FFPE procedure.

应用

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。

辅助数据和图表

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

资源

Sample to Insight solutions for successful molecular analysis

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

Critical factors for molecular analysis of FFPE samples

Download Safety Data Sheets for QIAGEN product components.

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