PAXgene Tissue RNA/miRNA Kit – Total RNA Purification

从固定和稳定于PAXgene Tissue Containers的组织中纯化miRNA和总RNA

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PAXgene Tissue RNA/miRNA Kit (50)

Cat. No. / ID: 766134

For 50 RNA preps: PAXgene RNA MinElute Spin Columns, PAXgene Shredder Spin Columns, Processing Tubes, Microcentrifuge Tubes, Carrier RNA, RNase-Free DNase, and RNase-Free Buffers; to be used in conjunction with PAXgene Tissue Containers

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仅作研究用途。不用于诊断程序。任何表述或表示都并非旨在提供诊断、预防或治疗疾病的信息。

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特点

固定、稳定、纯化的整体系统
高效纯化miRNA和总RNA
从组织中抽提高品质的miRNA
保持组织形态

产品详情

PAXgene Tissue miRNA Kit用于从固定和稳定于PAXgene Tissue Containers的组织中纯化总RNA，包括大于18 nt的RNA，纯化基于离心柱形式的硅胶膜RNA纯化技术。固定和储存于PAXgene Tissue Containers的组织样本可以是石蜡包埋的，用于病原体研究和后续的RNA、miRNA和/或DNA纯化。配合容器，该试剂盒为分子分析提供从采样、固定和稳定到高品质miRNA和总RNA纯化的完整解决方案。

绩效

采集器和试剂盒配合使用，构成分子分析中组织采集、固定和稳定，高品质RNA纯化，包括miRNA的分析完整解决方案（参见"Efficient purification of miRNA from fixed tissue stored in PAXgene Tissue Containers"）。

使用PAXgene Tissue miRNA Kit纯化的总RNA纯度非常高。将基因组DNA污染降至最低，纯化所得RNA可直接用于下游分析，无可检测到PCR抑制物。该试剂盒可分离出所有大于18nt的RNA分子。

原理

当前传统组织学中的组织固定方法对于分子分析来说应用是有限的。含有福尔马林的固定剂与生物分子交联，修饰核酸和蛋白质。在组织固定、储存和处理过程中，交联导致核酸降解。因为无法完全去除交联，其引起的化学修饰将限制敏感的下游应用，如定量PCR或RT-PCR。为了实现对同一样本进行分子病理检测和传统病理检测，需要一种方法在固定分子的同时保存组织形态。

PreAnalytiX研制了PAXgene Tissue System以满足这些需求。该系统包括组织采集装置（PAXgene Tissue Container用于采集、稳定、储存及运输人类样本组织）以及用于纯化总RNA、DNA或miRNA的试剂盒。PAXgene Tissue Containers可进行组织固定，用于组织病理学研究；也可从同一样本中纯化高纯度核酸，用于分子分析。固定和稳定方法保持了组织形态学，福尔马林固定组织中同时存在没有破坏性交联和降解的完整核酸。

为了纯化总RNA，包括miRNA，该系统需要应用PAXgene Tissue Containers采集和稳定组织，然后应用PAXgene Tissue miRNA Kit分离和纯化RNA。

程序

PAXgene Tissue Containers是预装有2种试剂的双腔容器。PAXgene Tissue Fix快速浸润并固定组织。固定之后，将其从PAXgene Tissue Fix中移出，转移到同一容器中含有PAXgene Tissue Stabilizer稳定液的另外一个腔内。组织被储存在PAXgene Tissue Stabilizer稳定液中，其核酸和组织形态可在室温下稳定3–7天，在2–8℃下可稳定2–4周，不同组织的保存时间不同。储存于-15–-30℃时，组织形态和核酸完整性可能稳定至少26个月。

固定了的样本可包埋于石蜡中，用于组织学研究。从PAXgene Tissue固定的组织样本中纯化核酸可在石蜡包埋之前或之后进行。

PAXgene Tissue miRNA Kit提供3种不同操作方案，用于从PAXgene Tissue Containers固定和稳定的组织中提取总RNA，包括小于200nt的RNA分子，例如5.8S rRNA、5S rRNA、tRNAs和miRNAs。经优化的结合和洗涤条件确保纯化小至18nt的RNA分子。作为先决条件，组织必须固定并稳定于PAXgene Tissue Containers中。

在结合缓冲液、Buffer TM1中破碎并匀质化组织样品。离心之后去除细胞碎片，在裂解液中添加乙醇确保可结合所有18nt以上的RNA。然后将样品放至PAXgene RNA MinElute离心柱上，总RNA结合到膜上，污染物被有效洗脱。在两次洗涤步骤之间，用DNase I处理膜，去除少量结合的DNA。洗涤之后，用低盐洗脱液洗脱包含miRNA的RNA，并加热使RNA变性（参见"The PAXgene Tissue miRNA procedure"）。

在PAXgene Tissue Fix和PAXgene Tissue Stabilizer中固定和贮存组织样品的条件是使用动物组织确定的。

应用

纯化所得miRNA和总RNA可即用于各种下游应用：

Northern印迹分析
RT-PCR和定量real-time RT-PCR
微阵列分析

辅助数据和图表

Efficient purification of miRNA from fixed tissue stored in PAXgene Tissue Containers.

Tissues from different organs were fixed for 3 hours in PAXgene Tissue Fix, transferred to PAXgene Tissue Stabilizer, and stored for 24 hours at room temperature. After processing, total RNA was purified from sections of paraffin-embedded tissue using the PAXgene Tissue RNA Kit (RNA/miRNA). miRNA-enriched fractions (200 nt) were also isolated from sections of the same tissue blocks using the PAXgene Tissue miRNA Kit (Enriched miRNA). Purified RNA was used as a template in quantitative, real-time RT-PCR assays for the miRNA miR-10a using the miScript Reverse Transcription Kit and the miScript SYBR^® Green PCR Kit.

Specifications

Features	Specifications
Time per run	Varies
Applications	PCR, qPCR, real-time PCR
Sample amount	25–100 mg
Elution volume	45–70 µl
Processing	Manual (centrifugation or vacuum)
Main sample type	Tissue samples
Yield	Varies
Technology	Silica technology
Format	96-well plate

资源

Groelz et al., BRN Symposium 2012

Groelz et al., ECP 2012

Groelz et al., BRN Symposium 2011

Groelz et al., 3rd Annual Oncology Biomarkers 2011

Groelz et al., AMP 2009

Groelz et al., ISBER 2012

Groelz et al., AMP 2008

Groelz et al., AACR 2010

Groelz et al., AMP 2009

Groelz et al., ECP 2014

Hesse et al., AACR-NCI-EORTC 2011

Groelz et al., AMP 2014

Moving toward excellence and standardization in tissue collection and fixation

Two worlds in one sample

Download Safety Data Sheets for QIAGEN product components.

Publications

A Novel Approach to High-Quality Postmortem Tissue Procurement: The GTEx Project.

Carithers LJ; Ardlie K; Barcus M; Branton PA; Britton A; Buia SA; Compton CC; DeLuca DS; Peter-Demchok J; Gelfand ET; Guan P; Korzeniewski GE; Lockhart NC; Rabiner CA; Rao AK; Robinson KL; Roche NV; Sawyer SJ; Segrè AV; Shive CE; Smith AM; Sobin LH; Undale AH; Valentino KM; Vaught J; Young TR; Moore HM; GTEx Consortium;

Biopreserv Biobank; 2015; 13 (5):311-9 2015 Oct PMID:26484571

Surgical Specimens of Colorectal Cancer Fixed with PAXgene Tissue System Preserve High-Quality RNA.

Hara K; Watanabe A; Matsumoto S; Matsuda Y; Kuwata T; Kan H; Yamada T; Koizumi M; Shinji S; Yamagishi A; Ishiwata T; Naito Z; Shimada T; Uchida E;

Biopreserv Biobank; 2015; 13 (5):325-34 2015 Oct PMID:26484572

Comprehensive DNA Methylation and Extensive Mutation Analyses of HER2-Positive Breast Cancer.

Yamaguchi T; Mukai H; Yamashita S; Fujii S; Ushijima T;

Oncology; 2015; 88 (6):377-84 2015 Jan 14 PMID:25591616

Next-gen tissue: preservation of molecular and morphological fidelity in prostate tissue.

Gillard M; Tom WR; Antic T; Paner GP; Lingen MW; VanderWeele DJ;

Am J Transl Res; 2015; 7 (7):1227-35 2015 Jul 15 PMID:26328007

Assessment of PAXgene Fixation on Preservation of Morphology and Nucleic Acids in Microdissected Retina Tissue.

Liu Y; Edward DP;

Curr Eye Res; 2016; 42 (1):104-110 2016 Jul 13 PMID:27409982

FAQ

You can find additional information relating to the PreAnalytiX PAXgene products on the PreAnalytiX website .

FAQ ID - 3515

For the isolation of microRNA (miRNA) specifically, we have developed the miRNeasy Mini Kit and the miRNeasy 96 Kit for isolation from cells and tissues. We also have the PAXgene Blood miRNA and Tissue miRNA kits for isolation from blood stored in PAXgene Blood RNA tubes and PAXgene Tissue Containers, respectively. Other miRNA isolation supplementary protocols can also be found by searching our comprehensive protocols at http://www.qiagen.com/literature/default.aspx?WT.svl=m.

FAQ ID -115

To prevent biomolecule degradation during processing, dehydration must begin with at least 70–100% ethanol. We recommend using low-melting paraffin (melting point ≤56°C) and do not incubate samples in liquid paraffin for more than 3 hours.

Processing protocols for the PAXgene Tissue System are listed in the appendices of the PAXgene Tissue Container Product Circular and PAXgene Tissue FIX Container (50 ml) Product Circular.

FAQ ID -2523

The PAXgene Tissue fixation and stabilization chemistry is free of cross-linking reagents. RNA purified from PFPE and PFCE is free of chemical modifications and performs similarly or identically to RNA isolated from frozen tissue. For examples of the correlation of gene expression levels in snap frozen tissue, FFPE, and PFPE, see Figure 4 in Groelz et al., Exp Mol Pathol. 2013 Feb; 94(1) and Figure 3 in Viertler et al., J Mol Diagn. 2012 Sep; 14(5).

FAQ ID -2538

The PAXgene Tissue DNA, RNA, and miRNA Kits are based on proven QIAGEN technologies. Nucleic acids isolated with these kits are generally of high purity.

On average, measurements of the A₂₆₀/A₂₈₀ ratio for DNA purified with the PAXgene Tissue DNA Kit are >1.7, and ratios for RNA and miRNA purified with the PAXgene Tissue RNA or miRNA Kits are both >1.8.

For examples of RNA purity see Technical Note "Yield, purity, and integrity of RNA purified from PAXgene Tissue fixed, paraffin-embedded (PFPE) rat tissue" under the Product Resources tab.

FAQ ID -2533

Yes. Supplementary protocols for generating sections from PAXgene Tissue fixed, Paraffin-embedded (PFPE) and PAXgene Tissue fixed, cryo-embedded (PFCE) tissue blocks for manual and laser microdissection are available under the Product Resources tab.

FAQ ID -2531