PAXgene Tissue RNA/miRNA Kit

For purification of microRNA and total RNA from tissues fixed and stabilized in PAXgene Tissue Containers

Features

  • Integrated fixation, stabilization, and purification
  • Effective purification of miRNA and total RNA
  • High-quality miRNA from tissues
  • Preserved tissue morphology
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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
$579.00
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For Research Use Only. Not for use in diagnostics procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

Tissue samples fixed and stored in PAXgene Tissue Containers can be paraffin-embedded and used for pathological studies as well as for subsequent purification of miRNA, RNA, and/or DNA. The PAXgene Tissue miRNA Kit provides purification of total RNA, including RNA from approximately 18 nucleotides, from tissues fixed and stabilized in PAXgene Tissue Containers. Purification is carried out using silica-based RNA purification technology in a spin-column format. Used with the containers, the kit provides a complete preanalytical solution for collection, fixation, and stabilization through to purification of high-quality miRNA and total RNA for molecular analysis.

Performance

Together the container and kit provide a complete preanalytical solution for collection, fixation, and stabilization of tissue, and for purification of high-quality RNA, including miRNA, for molecular analysis (see figure " Efficient purification of miRNA from fixed tissue stored in PAXgene Tissue Containers").

Total RNA purified using the PAXgene Tissue miRNA Kit is highly pure. Genomic DNA contamination is minimized, and purified RNA is ready to use in downstream applications with no detectable PCR inhibition. All RNA molecules longer than 18 nucleotides are purified.

See figures

Principle

Current tissue fixation methods used in traditional histology are of limited use for molecular analysis. Fixatives that contain formaldehyde cross-link biomolecules and modify nucleic acids and proteins. During tissue fixation, storage, and processing, cross-links lead to degradation of nucleic acids. Since cross-links can not be removed completely, the resulting chemical modifications can cause inhibition in sensitive downstream applications such as quantitative PCR or RT-PCR. In order to enable both molecular and traditional pathology testing from the same specimen, a method is needed for stabilization of molecular content and preservation of morphology.

PreAnalytiX has developed the PAXgene Tissue System to meet such needs. The system consists of a tissue collection device (the PAXgene Tissue Container for collection, stabilization, storage, and transportation of human tissue specimens) and kits for purification of miRNA, total RNA, or DNA. PAXgene Tissue Containers provide tissue fixation for histopathology studies and enable purification of high-quality nucleic acids from the same sample for molecular analysis. The fixation and stabilization method preserves tissue morphology and the integrity of nucleic acids without destructive cross-linking and degradation found in formalin-fixed tissues.

For purification of total RNA including miRNA, the system requires the use of PAXgene Tissue Containers for tissue collection and stabilization, followed by RNA isolation and purification using the PAXgene Tissue miRNA Kit.

Procedure

PAXgene Tissue Containers are dual-chamber containers prefilled with 2 reagents. PAXgene Tissue Fix rapidly penetrates and fixes the tissue. After fixation, the tissue is removed from the PAXgene Tissue Fix and transferred to PAXgene Tissue Stabilizer in the second chamber of the same container. When the tissue is stored in PAXgene Tissue Stabilizer, nucleic acids and morphology of the tissue sample are stable for a minimum of 3 and a maximum of 7 days at room temperature or for up to 8 weeks at 2–8°C, depending on the type of tissue. Storage at –15 to –30°C is also possible for at least 26 months without any negative effects on the morphology of the tissue or the integrity of the nucleic acids.

Stabilized samples can be embedded in paraffin for histological studies. Nucleic acids can be isolated from the PAXgene Tissue fixed, paraffin-embedded (PFPE) samples either before or after embedding in paraffin.

The PAXgene Tissue miRNA Kit provides 3 protocols for purification of total RNA from tissue fixed and stabilized in PAXgene Tissue Containers, including RNA molecules smaller than 200 nucleotides, such as 5.8S rRNA, 5S rRNA, tRNAs, and miRNAs. Optimized binding and washing conditions ensure the purification of RNA molecules as small as 18 nucleotides. As a prerequisite, the tissue must be fixed and stabilized in PAXgene Tissue Containers.

Disruption and homogenization of the tissue sample is performed in the binding buffer, Buffer TM1. After a centrifugation step to remove residual cell debris, ethanol 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 the total RNA binds to the membrane and contaminants are efficiently washed away. Between the first and second wash steps, 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 (see figure " The PAXgene Tissue miRNA procedure").

Specifications for fixation and storage conditions in PAXgene Tissue Fix and PAXgene Tissue Stabilizer were determined using animal tissues.

See figures

Applications

The purified miRNA and total RNA is ready to use in a wide range of downstream applications, including:

  • Northern blot analysis
  • RT-PCR and quantitative, real-time RT-PCR
  • Microarray analysis

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsNorthern blot analysis, RT-PCR and quantitative, real-time RT-PCR, microarray analysis
Sample amount4 x 15 x 15 mm
Elution volume14–40 µl
ProcessingManual (centrifugation)
Time per run60 min + 15 min incubation/8 samples
Main sample typeHuman tissue
YieldDepends on tissue type and starting material (fixed or PFPE*) * PAXgene Fixed Paraffin Embedded.
TechnologySilica technology
FormatSpin column

Resources

Scientific Posters (12)
Brochures & Guides (3)
Moving toward excellence and standardization in tissue collection and fixation

 

Two worlds in one sample
Kit Handbooks (1)

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

Where can I find additional information for PreAnalytiX PAXgene products?
You can find additional information relating to the PreAnalytiX PAXgene products on the PreAnalytiX website .
FAQ ID - 3515
What can I use to isolate RNA smaller than 200 nucleotides?

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
Is a special processing protocol needed?

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
What is the RT-PCR performance of RNA purified from PAXgene Tissue fixed, paraffin-embedded (PFPE) and PAXgene Tissue fixed, cryo-embedded (PFCE) tissue compared to RNA from snap frozen or formalin-fixed, paraffin-embedded (FFPE) tissue?
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
What is the purity of nucleic acids extracted with the PAXgene Tissue kits?

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
Is it possible to microdissect PAXgene Tissue fixed, paraffin-embedded (PFPE) and PAXgene Tissue fixed, cryo-embedded (PFCE) tissues?
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