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PAXgene Tissue RNA/miRNA Kit

For isolation and 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 from tissue with preserved morphology
  • Protocols for paraffin-embedded or paraffin-free tissues
  • Minimal genomic DNA contamination

Feature

Specification

Format

Spin column

Technology

Silica membrane

Main sample type

Human tissue

Sample amount

  • Up to 10 mg from PF* tissue
  • 2–5 sections 10 µm thick from PFPE** tissue with a maximum
  • size of 15 x 15 mm

Elution volume

14–40 µl

Time per run

  • 70 min + 15 min incubation/8 samples
    (120 min for fibrous tissues)

Processing

Manual: centrifugation

* PAXgene Tissue-fixed
** PAXgene Tissue-fixed, paraffin-embedded


Intended Use

The PAXgene Tissue RNA/miRNA Kit is intended for 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 additionally includes the PAXgene Tissue FIX Container and the PAXgene Tissue STABILIZER Concentrate for the collection, fixation, stabilization and storage of tissue samples.

For research use only. Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease. The performance characteristics of this product have not been fully established.

Order information

Product

Catalog Nr.

Price

PAXgene Tissue RNA/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 FIX Containers + PAXgene Tissue STABILIZER reagent.

766134
578 USD
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Details

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, tRNAs and miRNAs. Studies have shown that miRNA purified from PFPE tissue samples is reliably quantified, with high concordance to flash-frozen samples (Figure 1).

Principle

The PAXgene Tissue RNA/miRNA Kit enables purification of total RNA from tissues fixed and stabilized using the PAXgene Fixation and Stabilization products, 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 (Figure 2). 

Procedure

PAxgene Tissue-fixed (PF) or PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissue samples are disrupted 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 bound DNA. Total RNA, including miRNA, is then eluted in a low-salt elution buffer and denatured by heating (Figure 3).

The PAXgene Tissue RNA/miRNA Kit provides 2 protocols for purification of RNA, including miRNA, from different starting materials:

  • Sections of PFPE tissues
  • 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 (see Resources), including:

  • cDNA synthesis
  • Gene expression arrays
  • End-point RT-PCR
  • Quantitative RT-PCR
  • Detection and quantification of miRNA
  • RNA sequencing
     
RNA, including miRNA, 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 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, SCARNA17, SNORA73A; R2: coefficient of determination.Zoom image

Figure 1. High concordance of miRNA expression between total RNA isolated from PFPE and flash-frozen tissue.

RNA, including miRNA, purified from mirrored human breast cancer tissue fresh ...
SYBR Green real-time RT-qPCR was performed with 10 ng RNA from cryopreserved, formalin-fixed, paraffin-embedded (FFPE) or PAXgene Tissue-fixed, paraffin-embedded (PFPE) rat tissue (modified according to Groelz et al. (2013) Non-formalin fixative versus formalin-fixed tissue: a comparison of histology and RNA quality. Exp. Mol. Path. 94, 188). 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.Zoom image

Figure 2. 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, ...
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 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.Zoom image

Figure 3. The PAXgene Tissue RNA/miRNA Procedure.

Disruption and homogenization of the tissue sample is performed in binding buffer, ...

Resources

The PAXgene Tissue RNA/miRNA Kit Handbook is currently under revision. Please refer to the PAXgene Tissue miRNA Kit Handbook included in your kit for use instructions. For any questions, please contact us.

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For questions about the PAXgene Tissue RNA QIAcube protocol, please contact us.

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Simultaneous Purification of Genomic DNA and Total RNA, including miRNA, from Sections of PAXgene Tissue-fixed, Paraffin-embedded (PFPE) Tissue

82.6 KB

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Deparaffinization of PAXgene Tissue-fixed, Paraffin-embedded Tissue (PFPE) Sections with Deparaffinization Solution

614.1 KB

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Purification of Total RNA from Sections of PAXgene Tissue-fixed, Cryo-embedded (PFCE) Tissue Placed Directly into a Microcentrifuge Tube

589.1 KB

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Purification of Total RNA from Microdissected PAXgene Tissue-fixed, Paraffin-embedded and PAXgene Tissue-fixed, Cryo-embedded (PFCE) Tissues

718.0 KB

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Purification of Total RNA, including miRNA, from Sections of PAXgene Tissue-fixed, Cryo-embedded (PFCE) Tissue Placed Directly into a Microcentrifuge Tube

606.8 KB

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Purification of Total RNA, including miRNA, from Microdissected PAXgene Tissue-fixed, Paraffin-embedded (PFPE) and PAXgene Tissue-fixed, Cryo-embedded (PFCE) Tissues

662.9 KB

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Manual Processing of Tissue Specimens Treated with the PAXgene Tissue System

334.1 KB

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PAXgene Tissue System Brochure

1.9 MB

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Influence on RNA Yield and Integrity of Modifications to the Processing Protocol for PAXgene Tissue-fixed, Paraffin-embedded (PFPE) Rat Tissue

633.0 KB

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Morphology and RNA Preservation in PAXgene Tissue-fixed, Paraffin-embedded Tissue (PFPE) Stored at 18 Months at Different Temperatures

483.8 KB

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Simultaneous Preservation of RNA and Morphology in Tissue Samples Fixed with PAXgene Tissue Fix and Stored in PAXgene Tissue STABILIZER at –20°C or –80°C

1.3 MB

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Yield, Purity and Integrity of RNA Purified from PAXgene Tissue-fixed, Paraffin-embedded (PFPE) Rat Tissue

603.8 KB

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RNA Stability in Tissue Samples, Fixed and Stabilized with the PAXgene Tissue System, for up to 7 Days at 22°C or 2 Months at 4°C

524.9 KB

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Vacuum Sealing of Fixed and Stabilized Tissue with a FoodSaver Vaccum Sealer for Dry and Safe Transport

864.9 KB

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Influence of Formalin Contamination During Processing of PAXgene Tissue-fixed, Paraffin-embedded Tissue (PFPE) on RNA Yield, Integrity and Performance in Quantitative RT-PCR

661.1 KB

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Storage of Tissue in PAXgene Tissue STABILIZER: RNA and Morphology Preservation after 5 Years at –20 and 3 Years at –80°C (Groelz 2014)

887.7 KB

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RT-PCR Performance of RNA Obtained From Archived Formalin or PAXgene Tissue Fixed, Paraffin-embedded (FFPE and PFPE) Blocks of Tissue (Groelz 2014)

7.2 MB

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Stability of Nucleic Acids in Archived Formalin and PAXgene Tissue Fixed, Paraffin-embedded (FFPE and PFPE) Blocks of Tissue (Groelz 2014)

2.5 MB

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PAXgene vs. Formalin Fixed Tissue: A Comparison of Tissue Morphology and RNA Quality (Groelz 2012)

5.0 MB

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Preservation of Morphology and Biomolecules Within Tissue Stored for Three Years at –80°C in PAXgene Tissue Stabilizer Reagent (Groelz 2012)

890.5 KB

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PAXgene Tissue Fixation Technology for Simultaneous Preservation of Morphology and Biomolecules (Groelz 2012)

1.2 MB

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A New Tissue Fixative for Biomarker Discovery: Gene Expression and miRNA in PAXgene Tissue-fixed, Paraffin-embedded (PFPE) Colorectal Cancer (CRC) and Breast Cancer Tissue vs. Formalin-fixed, Paraffin-embedded (FFPE) Tissue (Groelz 2011)

413.4 KB

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PAXgene Tissue: A New Tissue Fixation Technology for Simultaneous Preservation of Morphology and Nucleic Acids (Groelz 2011)

542.1 KB

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Evaluation of the PAXgene Tissue System: Preservation of Morphology and Gene Expression in Human Melanoma (Hesse 2011)

973.5 KB

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Preservation of Gene Expression Profile and Histomorphology in Human Breast Tumor Tissue with the New PAXgene Tissue System (Groelz 2010)

750.8 KB

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Preservation of Histomorphology and Nucleic Acids in Human Breast Tumor TIssue with the New PAXgene Tissue System — A Study with Comparison to Formalin Fixation (Groelz 2009)

934.1 KB

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Long-term Storage of Tissue Specimens at –20°C to –80°C with Preservation of Morphology and Nucleic Acids Within Frozen Tissue (Groelz 2009)

676.9 KB

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New Fixation Technology for Simultaneous Preservation of Mophology and Nucleic Acids in Tissue (Groelz 2008)

516.6 KB

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Groelz, D., et al. (2018) Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues. PLoS ONE 13(9): e0203608.

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Wiethaler, M., et al. (2019) BarrettNET – a prospective registry for risk estimation of patients with Barrett's esophagus to progress to adenocarcinoma. Dis. Esophagus, doz024

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Yamazaki, M., et al. (2018) PAXgene-fixed paraffin-embedded sample is applicable to laser capture microdissection with well-balanced RNA quality and tissue morphology. J. Toxicol. Pathol. 31: 213.

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Liu, Y. and Edward, D.P. (2017) Assessment of PAXgene Fixation on Preservation of Morphology and Nucleic Acids in Microdissected Retina Tissue. Curr. Eye Res. 42, 104. E-published in 2016. doi: 0.3

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Ruusuvuori, P., et al. (2016) Feature-based analysis of mouse prostatic intraepithelial neoplasia in histological tissue sections. J. Pathol. Inform. 7, 5. doi: 10.4103/2153-3539.175378

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Loibner, M., et al. (2016) Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative. PLoS One 11, e0151383. doi: 10.1

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Oberauner-Wappis, L., et al. (2016) Protocol for HER2 FISH determination on PAXgene-fixed and paraffin-embedded tissue in breast cancer. Int. J. Exp. Pathol. 97, 202. doi: 10.1111/iep.12185

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Korenkova, V., et al. (2016) The focus on sample quality: Influence of colon tissue collection on reliability of qPCR data. Sci. Rep. 6, 29023. doi: 10.1038/srep29023

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Mathieson, W., et al. (2016) A Critical Evaluation of the PAXgene Tissue Fixation System:  Morphology, Immunohistochemistry, Molecular Biology, and Proteomics. Am. J. Clin. Pathol. 146, 25. doi: 0.

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Hara, K., et al. (2015) Surgical Specimens of Colorectal Cancer Fixed with PAXgene Tissue System Preserve High-Quality RNA. Biopreserv. Biobank. 13, 325. doi: 10.1089/bio.2014.0101

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Gillard, M., et al. (2015) Next-gen tissue: preservation of molecular and morphological fidelity in prostate tissue. Am. J. Transl. Res. 7, 1227.

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Keen, J.C. and Moore, H.M. (2015) The Genotype-Tissue Expression (GTEx) Project: Linking Clinical Data with Molecular Analysis to Advance Personalized Medicine. J. Pers. Med. 5, 22. doi: 0.3390/jp

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Carithers, L.J., et al. (2015) A Novel Approach to High-Quality Postmortem Tissue Procurement: The GTEx Project. Biopreserv. Biobank. 13, 311. doi: 10.1089/bio.2015.0032

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PAXgene Tissue RNA/miRNA Kit

Purification and quality of biomolecules from PAXgene Tissue-treated samples

1. Is it possible to use formalin-fixed, paraffin-embedded (FFPE) kits and protocols to isolate biomolecules from PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissues?
No. Procedures developed for the extraction of biomolecules from FFPE tissues include prolonged proteinase K digestion and heating steps to remove chemical modifications introduced by formalin. Since the PAXgene Tissue System does not chemically modify biomolecules, these steps are not necessary and, in fact, lead to degradation of biomolecules. Instead, use dedicated PAXgene Tissue Kits and supplementary protocols for extraction of biomolecules from PAXgene Tissue-treated samples.


2. What is the purity of nucleic acids extracted with the PAXgene Tissue Kits?
The PAXgene Tissue DNA and RNA/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, including miRNA, purified with the PAXgene Tissue RNA/miRNA Kit are >1.8.

3. What is the average RNA yield, including miRNA, from PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissues?    
RNA yield, including miRNA, depends on several parameters, such as tissue type, time from resection until fixation, fixation time, processing protocol used and age and storage conditions of the PFPE block.

In a study with PFPE tissue sections (area: 100 mm²;  thickness: 10 µm) median RNA yield from rat liver was 4.2 µg (n=58), from kidney 2.2 µg (n=58), from spleen 4.7 µg (n=58), from intestine 4.7 µg (n=58) and from lung 0.9 µg (n=58). See the Technical Note "Yield, purity, and integrity of RNA purified from PAXgene Tissue fixed, paraffin-embedded (PFPE) rat tissue" under Resources.    

4. How well is RNA integrity preserved in PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissues?  
Similar to yield, RNA integrity depends on several parameters, such as tissue type, time from resection until fixation, fixation time, processing protocol and age and storage conditions of the PFPE block. For examples of RNA integrity values from rat tissues under ideal workflow conditions, see Groelz et al. 2013under References. For examples of RNA integrity from clinical samples, see Viertler et al. 2012.

5. What are the yield and integrity of nucleic acids isolated from blocks of PAXgene Tissue-fixed, cryo-embedded (PFCE) tissues?    
DNA and RNA isolated from PFCE tissue specimens are of high quantity and quality, comparable to PFPE tissue.    


6. Are special kits and protocols required for the isolation of biomolecules from PAXgene Tissue fixed, cryo-embedded (PFCE) tissues?    
No. Regular PAXgene Tissue Kits can be used for the isolation of RNA, miRNA and DNA from PFCE tissue. Supplementary protocols developed specifically for the extraction of biomolecules from PFCE samples are available under Resources.


7. Which kits and protocols can be used for the isolation of nucleic acids from microdissected PAXgene-Tissue fixed, paraffin-embedded (PFPE) and PAXgene Tissue-fixed, cryo-embedded (PFCE) specimens?    
Regular PAXgene Tissue Kits can be used for the isolation of RNA, miRNA and DNA from microdissected PFPE and PFCE tissue. Supplementary protocols developed specifically for the extraction of biomolecules from microdissected PFPE and PFCE samples are available under Resources.    


Molecular analysis of biomolecules purified from PAXgene Tissue-treated samples    

1. What is the RT-PCR performance of RNA purified from PAXgene Tissue-fixed, paraffin-embedded (PFPE) and PAXgene Tissue-fixed, cryo-embedded (PFCE) tissues compared to RNA from snap-frozen or formalin-fixed, paraffin-embedded (FFPE) tissues?    
RNA, including miRNA, 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. 2013and Figure 3 in Viertler et al. 2012 under Resources.

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