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PAXgene Tissue RNA/miRNA Kit

For purification of total RNA, including miRNA, from tissues fixed and stabilized using the PAXgene Tissue System
  • Effective purification of high and low molecular weight RNA
  • Preserved tissue morphology
  • Protocols for paraffin-embedded and paraffin-free samples
  • Minimal genomic DNA contamination
The PAXgene Tissue RNA/miRNA Kit is intended fro the purification of total RNA, including miRNA, from PAXgene Tissue-fixed (PF) and PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissue samples. The kit is intended to be used as part of the PAXgene Tissue System, which also includes the PAXgene Tissue FIX Container and PAXgene Tissue STABILIZER Concentrate for the collection, fixation, stabilization and storage of tissue samples.
Cat No./ID: 766134
PAXgene Tissue miRNA Kit (50)
€476.00
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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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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
Total RNA isolated with the PAXgene Tissue RNA/miRNA Kit is highly pure. Genomic DNA contamination is minimized and the purified RNA is ready to be used in downstream applications with no detectable inhibition of PCR. The resulting eluate includes smaller RNA molecules, such as 5.8S rRNA, 5S rRNA, tRNA and miRNAs. Studies have shown that the miRNA purified from PFPE tissue samples is reliably quantified, with high concordance to flash-frozen samples (see High concordance of miRNA expression between total RNA isolated from PFPE and flash-frozen tissue).
Principle
The PAXgene Tissue RNA/miRNA Kit enables purification of total RNA from tissues fixed and stabilized using the PAXgene fixation and stabilization reagents, which preserve tissue morphology and biomolecule integrity by avoiding destructive crosslinking and degradation found in formalin-fixed tissues. The purified RNA and miRNA have no inhibitory chemical modifications and thus, can be used in sensitive downstream applications (see RNA purified without chemical modification from PFPE tissue using the PAXgene Tissue RNA/miRNA Kit).
Procedure

PAXgene Tissue-fixed (PF) or PAXgeen Tissue-fixed, paraffin-embedded (PFPE) tissue samples are distrupted and homogenized in binding buffer. After centrifugation to remove cellular debris, optimal conditions are created for binding of RNA molecules to the silica membrane. Contaminants are then washed away and DNase I treatment removes any trace amounts of DNA. Total RNA, including miRNA, is then eluted in a low-salt elution buffer and denatured by heating (see The PAXgene Tissue RNA/miRNA Procedure).

The PAXgene Tissue RNA/miRNA Kit provides 2 protocols for purification of RNA, including miRNA, from different starting materials: from sections of PFPE tissues and from PF tissue samples (without paraffin embedding).

Applications

Total RNA and miRNA purified with the PAXgene Tissue RNA/miRNA Kit is ready to be used in a range of downstream research applications, including:

  • cDNA synthesis
  • Gene expression arrays
  • End-point RT-PCR
  • Quantitative RT-PCR
  • Detection and quantification of miRNA
  • RNA sequencing

 

Features
Specifications
Applications Northern blot analysis, RT-PCR and quantitative, real-time RT-PCR, microarray analysis
Elution volume 14–40 µl
Format Spin column
Main sample type Human tissue
Processing Manual (centrifugation)
Sample amount 4 x 15 x 15 mm
Technology Silica technology
Time per run 60 min + 15 min incubation/8 samples
Yield Depends on tissue type and starting material (fixed or PFPE*) * PAXgene Fixed Paraffin Embedded.

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Brochures & Guides (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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Scientific Posters (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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Kit Handbooks (1)
For isolation and purification of total RNA, including miRNA, from tissue samples stabilized in PAXgene Tissue Containers
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References
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