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PAXgene Tissue miRNA Kit

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

  • 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

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。

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Cat No./ID: 766134
PAXgene Tissue miRNA Kit (50)
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

仅限于科研应用,不适用于诊断,不为疾病的诊断、预防或治疗提供参考信息。


Product Details

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The PAXgene Tissue RNA/miRNA Procedure.

Disruption and homogenization of the tissue sample is performed in binding buffer, Buffer TM1. After centrifugation to remove residual cell debris, isopropanol is added to the lysate to provide appropriate binding conditions for all RNA molecules 18 nucleotides and longer. The sample is then applied to a PAXgene RNA MinElute spin column, where total RNA binds to the membrane and contaminants are efficiently washed away. Between the first and the second wash step, the membrane is treated with DNase I to remove trace amounts of bound DNA. After the wash steps, RNA, including miRNA, is eluted in a low-salt elution buffer and denatured by heating.

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High concordance of miRNA expression between total RNA isolated from PFPE and fresh-frozen tissue.

RNA, including miRNA, was purified from mirrored human breast cancer tissue fresh frozen in liquid nitrogen using the QIAGEN miRNeasy Kit, or from sections of PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissue using the PAXgene Tissue RNA/miRNA Kit. Shown is a scatterplot of CT values from different single miRNA-specific RT-qPCR assays using the QIAGEN miScript System: miR9, -10a, -10b, -29a, -103, -125b, -143, -145, -192 and miScript PCR controls RNUA1, RNU5A, RNU6B, SNORD25, SCARNA19, SNORA73A; R2: coefficient of determination.

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RNA purified without chemical modification from PFPE tissue using the PAXgene Tissue RNA/miRNA Kit.

SYBR Green real-time RT-qPCR was performed with 10 ng RNA from cryopreserved, formalin-fixed, paraffin-embedded (PPFE) or PAXgene Tissue-fixed, paraffin-embedded (PFPE) rat tissue (modified according to Groelz et al. 2013). Depicted are the average delta-CT values (delta-CT = CT[FFPE] – CT[cryo] or delta-CT = CT[PFPE] – CT[cryo]) from 6 different assays with amplicons ranging from 109 to 465 bp.

Performance

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。
Principle

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。
Procedure

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。

Applications

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

Specifications

Features
Specifications
应用 PCR, qPCR, real-time PCR
洗脱体积 45–70 µl
规格 96-well plate
主要样本类型 Tissue samples
处理 Manual (centrifugation or vacuum)
样本量 25–100 mg
技术 Silica technology
Time per run Varies
产量 Varies

Product Resources

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产品介绍与指南 (4)
Two worlds in one sample
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For isolation and purification of total RNA, including miRNA, from tissue samples fixed and stabilized using the PAXgene Tissue System

 


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Moving toward excellence and standardization in tissue collection and fixation

 


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学术海报 (12)
Hesse et al., AACR-NCI-EORTC 2011

 


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Groelz et al., ECP 2012

 


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Groelz et al., 3rd Annual Oncology Biomarkers 2011

 


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Groelz et al., BRN Symposium 2011 

 


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Groelz et al., AMP 2014

 


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Groelz et al., ISBER 2012

 


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Groelz et al., AACR 2010

 


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Groelz et al., BRN Symposium 2012 

 


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Groelz et al., AMP 2009 

 


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Groelz et al., ECP 2014

 


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Groelz et al., AMP 2008 

 


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Groelz et al., AMP 2009

 


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试剂盒操作手册 (1)
For isolation and purification of total RNA, including miRNA, from tissue samples stabilized in PAXgene Tissue Containers
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参考文献
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