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PAXgene Tissue STABILIZER Concentrate

For stabilization of tissue specimens fixed in PAXgene Tissue FIX

  • Preservation of both morphology and biomolecules
  • Tissue can be stored and archived for later processing
  • RNA, miRNA, DNA and/or proteins from one sample
  • Can be used to fill a tissue processor at position one

Feature

Specification

Bottle size

500 ml filled with 150 ml PAXgene Tissue STABILIZER Concentrate

Additive

PAXgene Tissue STABILIZER Concentrate
Note: must be used with PAXgene Tissue FIX Container

Quantity

8 bottles (makes 4 liters of PAXgene Tissue STABILIZER Reagent)

Length of stabilization

  • up to 7 days at room temperature (15–25°C)
  • up to 4 weeks at 2–8°C*
  • long-term at –20°C or –80°C**

Archiving options for PFPE***

  • short-term at room temperature or 2–8°C
  • long-term at –20°C or –80°C**

*   Storage at 2–8°C for more than 4 weeks must be
    validated for each tissue type
**  Long-term storage studies are ongoing
*** PAXgene Tissue-fixed, paraffin-embedded


Intended Use

The PAXgene Tissue STABILIZER Concentrate is intended for the stabilization and storage of tissue specimens. This stabilizing agent is intended to be used as part of the PAXgene Tissue System and must be used in conjuction with the PAXgene Tissue FIX Container (see Procedure below). The PAXgene Tissue System additionally includes the PAXgene Tissue RNA/miRNA Kit for the isolation of total RNA, including miRNA, and the PAXgene Tissue DNA Kit for DNA. Supplementary protocols are available for other applications, including purification of proteins.

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 STABILIZER Concentrate (150 ml)

8 bottles of 150 ml PAXgene Tissue Stabilizer Concentrate. Makes 4 liters of PAXgene Tissue STABILIZER Reagent for use with tissue specimens previously fixed in PAXgene Tissue FIX Containers.

765512
230 USD
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Performance

PAXgene Tissue STABILIZER Concentrate serves to stabilize and preserve tissue morphology and the integrity of biomolecules without destructive crosslinking and degradation found in formalin-fixed tissues (Figure 1 and Figure 2). While the stabilizing action of this agent is designed for tissues that have been previously fixed in PAXgene Tissue FIX, appropriately diluted PAXgene Tissue STABILIZER Concentrate can be used in paraffin-embedding with a tissue processor.

Enhanced storage

When tissues previously fixed in PAXgene Tissue FIX are stored in PAXgene Tissue STABILIZER, nucleic acids, proteins and morphology are stable for up to 7 days at room temperature (15–25°C) or 4 weeks at 2–8°C. Tissue samples can be stored in the PAXgene Tissue STABILIZER for longer periods at –20°C (–15°C to –30°C) or –80°C (–65°C to –90°C) without negative effects on the morphology of the tissue or the integrity of analytes.

Note: Specifications for storage conditions in PAXgene Tissue STABILIZER were determined using animal tissues. Storage at 2–8°C for more than 4 weeks must be validated for each tissue type.

Note: For storage at –20°C (–15°C to –30°C) or –80°C (–65°C to –90°C), use cryogenic vials with screw caps filled with PAXgene Tissue STABILIZER. For safety reasons, note that PAXgene Tissue STABILIZER contains 70% ethanol by volume.

High-quality biomolecule stabilization

Nucleic acids and proteins of tissues stored in PAXgene Tissue STABILIZER or as PAXgene Tissue-fixed, paraffin embedded (PFPE) samples are stabilized and can be purified using the corresponding PAXgene Tissue Kit for RNA and miRNA or DNA. Use the Qproteome FFPE Tissue Kit for purification of proteins. Purified biomolecules are of high quality (Figure 3) and unmodified (Figure 4). The purified biomolecules are highly suited for a range of demanding downstream applications (Figure 5Figure 6  and Figure 7).

Principle

The formalin-free PAXgene fixation and stabilization products can be used as an alternative to traditional histology methods for tissue fixation and analysis because they enable comparable processing of tissue samples without the crosslinking and degradation observed in formalin-based systems. The PAXgene Tissue STABILIZER enables stabilization of tissue specimens previously fixed in a PAXgene Tissue FIX Container. The system facilitates preservation of tissue morphology and stabilization of biomolecules so that histological and molecular analyses, including RT-PCR, qPCR and next-generation sequencing, can be performed on the same tissue specimen.

Procedure

PAXgene Tissue STABILIZER Concentrate is diluted with ethanol to make PAXgene Tissue STABILIZER Reagent, which is designed to be used with tissues that have been fixed in PAXgene Tissue FIX. After fixation of tissue samples with the PAXgene Tissue FIX Containers for 2–72 hours, the fixative in the container is removed and replaced by PAXgene Tissue STABILIZER. Stabilized samples can be stored long-term in the STABILIZER or embedded in paraffin for histological studies. Nucleic acids and proteins can be isolated from the stabilized samples before or after embedding in paraffin. PAXgene Tissue STABILIZER Reagent can also be used in paraffin embedding with a tissue processor (Figure 9).

Applications

PAXgene Tissue-fixed (PF) and PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissue samples can be used for (see ReferencesTissue Atlas):

 
Nucleic acids purified from PF and PFPE tissue samples can be used for demanding downstream applications (see ReferencesTechnical Notes), including:

  • RNA and miRNA profiling
  • Long-range and multiplex PCR
  • Next-generation sequencing

 
Proteins purified from PF and PFPE tissue samples can be used in a range of downstream applications (see ReferencesTechnical Notes), including:

  • Western blotting
  • Reverse-phase protein microarrays
  • MALDI imaging mass spectrometry
  • 2-D gel electrophoresis
Human tissue samples were divided into 2 sub-samples. One sub-sample was fixed with PAXgene Tissue FIX and the other half was fixed in neutral-buffered formalin. The fixed tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin. PFPE: PAXgene Tissue-fixed, paraffin-embedded; FFPE: formalin-fixed, paraffin-embedded.Zoom image

Figure 1. H&E staining with the PAXgene Tissue System gives results comparable to formalin-fixed tissue.

Human tissue samples were divided into 2 sub-samples. One sub-sample was fixed ...
Human palatine tonsil tissue was fixed in PAXgene Tissue FIX or with neutral-buffered formalin and embedded in paraffin. Primary antibodies to the indicated antigens were linked to a streptavidin-peroxidase conjugate by a biotinylated secondary antibody (LSAB method). Sections were counterstained with hematoxylin. PFPE: PAXgene Tissue-fixed, paraffin-embedded; FFPE: formalin-fixed, paraffin-embedded.Zoom image

Figure 2. Immunohistochemistry (IHC) staining with the PAXgene Tissue System gives comparable results to formalin-fixed tissue.

Human palatine tonsil tissue was fixed in PAXgene Tissue FIX or with ...
(A) Hematoxylin and eosin (H&E) staining of PAXgene Tissue-fixed, paraffin-embedded (PFPE) human colorectal cancer tissue and (B) DNA on agarose gel electrophoresis using 0.8 % TBE buffered gels with 200 ng genomic DNA isolated in triplicate from 5 cases (#1–5) of human PFPE colorectal cancer. M: markers.Zoom image

Figure 3. High-quality DNA from PFPE tissue with preserved morphology.

(A) Hematoxylin and eosin (H&E) staining of PAXgene Tissue-fixed, ...
Multiplex and long-range PCR of DNA from PAXgene Tissue-fixed, paraffin-embedded (PFPE) human colorectal cancer tissue (modified according to Viertler et al., (2012) A New technology for stabilization of biomolecules in tissues for combined histological and molecular analyses. J. Mol. Diagn. 14, 458). (A) Multiplex PCR of 8 different genomic DNA fragments ranging from 222 to 955 bp. (B) Long-range PCR of a 5 kb genomic DNA fragment.Zoom image

Figure 4. DNA without chemical modifications can be used for demanding downstream applications.

Multiplex and long-range PCR of DNA from PAXgene Tissue-fixed, paraffin-embedded ...
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 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, SCARNA17, SNORA73A; R2: coefficient of determination.Zoom image

Figure 5. High concordance of miRNA expression between total RNA isolated from PFPE and fresh-frozen tissue.

RNA, including miRNA, purified from mirrored human breast cancer tissue fresh ...
Non-malignant human uterus, breast, prostate and bladder specimens were divided into 3 samples and either flash-frozen in liquid nitrogen (cryo), PAXgene Tissue-fixed, paraffin-embedded (PFPE) or formalin-fixed, paraffin-embedded (FFPE). Proteins from cryo, PFPE, and FFPE tissues were extracted using the extraction buffer EXB Plus (Qproteome FFPE Tissue Kit; described in Ergin et al. 2010, Gündisch et al. 2013 and PAXgene Tissue supplementary protocols). SDS-PAGE and Western blot analysis were performed with 15 μg protein and the indicated antibodies. Data kindly provided by Karl-Friedrich Becker, Technical University of Munich, Germany.Zoom image

Figure 6. Detection of nondegraded, immunoreactive phosphoproteins from human PFPE tissue.

Non-malignant human uterus, breast, prostate and bladder specimens were divided ...
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). 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 7. 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, ...
Non-malignant human duodenum tissue was divided into 3 samples and either flash-frozen in liquid nitrogen (cryo), or prepared as PFPE or FFPE tissue. Proteins from cryo and PFPE tissue were extracted in 2D buffer (30 mM Tris-HCl pH8.8, 7 M urea, 2 M thiourea, 4 % CHAPS, 75 mM DTT) supplemented with protease inhibitor. Proteins from FFPE tissue were extracted in EXB Plus buffer supplemented with protease inhibitor, precipitated with acetone and resuspended in 2D buffer (as described in Gündisch et al. 2013). Samples (150 μg) were separated by 2-D PAGE. Data kindly provided by Karl-Friedrich Becker, Technical University of Munich, Germany.Zoom image

Figure 8. Protein extracted from PFPE tissue is suitable for two-dimensional gel electrophoresis.

Non-malignant human duodenum tissue was divided into 3 samples and either ...
Single-chamber container for fixation and stabilization of a single, larger or multiple, smaller tissue samples.Zoom image

Figure 9. The PAXgene Tissue FIX Container (50 ml) and PAXgene Tissue STABILIZER Workflow.

Single-chamber container for fixation and stabilization of a single, larger or ...
Twelve different PAXgene Tissue-fixed (PF) rat tissue types, including fibrous or fatty, DNA-rich and DNA-poor tissue, processed with the PAXgene Tissue DNA Kit on the QIAcube in one single run. DNA yield per 10 mg tissue.Zoom image

Figure 10. Yield of high-quality, high molecular weight DNA from 12 different PAXgene Tissue fixed (PF) tissue types, processed on the QIAcube.

Twelve different PAXgene Tissue-fixed (PF) rat tissue types, including fibrous or ...
Twelve different PAXgene Tissue-fixed (PF) rat tissue types, including fibrous or fatty, DNA-rich and DNA-poor tissue, processed with the PAXgene Tissue DNA Kit on the QIAcube in one single run. Ratio of absorbance at 260 and 280 nm.Zoom image

Figure 11. Absorbance ratio of high-quality, high molecular weight DNA from 12 different PAXgene Tissue fixed (PF) tissue types, processed on the QIAcube.

Twelve different PAXgene Tissue-fixed (PF) rat tissue types, including fibrous or ...
Twelve different PAXgene Tissue-fixed (PF) rat tissue types, including fibrous or fatty, DNA-rich and DNA-poor tissue, processed with the PAXgene Tissue DNA Kit on the QIAcube in one single run. DNA from each tissue type (300 ng) on agarose gel electrophoresis using 1% TBE buffered gels; M: markers.Zoom image

Figure 12. Agarose gel electrophoresis of high-quality, high molecular weight DNA from 12 different PAXgene Tissue fixed (PF) tissue types, processed on the QIAcube.

Twelve different PAXgene Tissue-fixed (PF) rat tissue types, including fibrous or ...
DNA yield from 3 sections (thickness 10 µm) each of 8 different PAXgene Tissue-fixed, paraffin-embedded (PFPE) rat tissue types processed in triplicate with the PAXgene Tissue DNA Kit on the QIAcube or manually.Zoom image

Figure 13. Comparison of manual and automated procedure: DNA yield from sections of PAXgene Tissue fixed, paraffin-embedded (PFPE) tissue.

DNA yield from 3 sections (thickness 10 µm) each of 8 different PAXgene ...
Ratio of absorbance at 260 and 280 nm from 3 sections (thickness 10 µm) each of 8 different PAXgene Tissue-fixed, paraffin-embedded (PFPE) rat tissue types processed in triplicate with the PAXgene Tissue DNA Kit on the QIAcube or manually.Zoom image

Figure 14. Comparison of manual and automated procedure: Absorbance ratio from sections of PAXgene Tissue fixed, paraffin-embedded (PFPE) tissue.

Ratio of absorbance at 260 and 280 nm from 3 sections (thickness 10 µm) each of 8 ...
Agarose gel electrophoresis from 3 sections (thickness 10 µm) each of 8 different PAXgene Tissue-fixed, paraffin-embedded (PFPE) rat tissue types processed in triplicate with the PAXgene Tissue DNA Kit on the QIAcube or manually. DNA from each replicate and tissue type (200 ng) on agarose gel electrophoresis using 1% TBE buffered gels; M: markers.Zoom image

Figure 15. Comparison of manual and automated procedure: Agarose gel electrophoresis from sections of PAXgene Tissue fixed, paraffin-embedded (PFPE) tissue.

Agarose gel electrophoresis from 3 sections (thickness 10 µm) each of 8 different ...

Resources

PAXgene Tissue STABILIZER Concentrate

Tissue fixation and stabilization chemistry


1. Which fixation method is used in the PAXgene Tissue System?


The PAXgene Tissue System uses an acidic and alcoholic fixation without formalin that does not result in crosslinking of biomolecules.

2. What is the composition of PAXgene Tissue FIX?
The PAXgene Tissue FIX fixation reagent contains alcohols and an acid, in addition to other stabilization agents.

3. What is the composition of PAXgene Tissue STABILIZER?
The PAXgene Tissue STABILIZER stabilization reagent contains alcohol and other stabilization agents. It is available in bulk as a concentrate.

4. Are the two reagents used in the PAXgene Tissue System obligatory?
The PAXgene Tissue System involves two processes: fixation and stabilization. PAXgene Tissue FIX provides rapid penetration and fixation that effectively stops all enzymatic activity throughout the tissue. The tissue can remain in the fixative for maximum 72 h. For long-term transportation and storage, PAXgene Tissue STABILIZER stops fixation and stabilizes the specimen.

Tissue fixation and stabilization

1. What is the maximum tissue size that can be fixed in a PAXgene Tissue FIX Container (50 ml)?
Up to 4 standard tissue cassettes, each containing tissue samples with a maximum size of 4 x 15 x15 mm, or alternatively, a single tissue sample with a maximum size of 20 x 20 x 20 mm can be placed into a PAXgene Tissue FIX Container. If using a larger tissue sample surrounded by fat (e.g., from a lymph node) or a capsule (e.g., from kidney, liver or spleen), partially cut into the tissue every 5 mm (lamination) to enhance permeation of the fixation reagent. If samples are larger than recommended, the fast and even penetration of fixation reagent is compromised. This may result in quality reduction of tissue morphology and integrity of nucleic acids.

2. How long is the fixation time?
Depending on tissue size specimen(s) must be fixed at room temperature (15–25°C) for a minimum of 2 h (for samples up to 4 x 15 x 15 mm), or for a minimum of 6 h (for samples up to 20 x 20 x 20 mm). Fixation should be stopped by transfer into PAXgene Tissue STABILIZER after a maximum of 72 hours fixation.

For biopsies with a thickness of 1 mm or less, fixation time can be reduced to 30–60 min.

3. Which conditions are recommended for the storage of tissues in PAXgene Tissue STABILIZER?
Depending on tissue type, standard storage conditions in PAXgene Tissue STABILIZER are up to 7 days at room temperature (15–25°C) or up to 4 weeks at 2–8°C. Storage at 2–8°C for more than 4 weeks must be validated for each tissue type. For longer storage, samples can be kept at–15°C to –30°C or –65°C to –90°C. Long-term storage studies are ongoing. For the latest results, see the poster "RNA and Morphology Preservation after 5 years at –20°C and 3 years at –80°C" under Resources.

Processing

1. Is it possible to use a standard processor – the kind used routinely for formalin-fixed samples – for dehydration and paraffin infiltration of PAXgene Tissue-treated samples?
Yes. All processors commonly used for formalin-fixed samples can be used to produce PAXgene Tissue-fixed, paraffin-embedded (PFPE) blocks of tissue.

We recommend keeping alcohol for processing PAXgene Tissue-treated samples separate from alcohol used for processing formalin-fixed samples for at least the first five positions/steps in the processing. With this precaution, it is possible to process PAXgene Tissue-fixed and formalin-fixed samples on the same instrument.

2. Is it possible to process formalin-fixed and PAXgene Tissue-fixed samples together in one run?
Parallel processing of formalin-fixed and PAXgene Tissue-fixed samples can lead to reduction of nucleic acid yield and integrity from PFPE samples through formalin contamination of reagents.

3. Is it necessary to clean a processor normally used for formalin-fixed tissue before using it with PAXgene Tissue-fixed tissue?
No. Special cleaning is not required. However, when processing PAXgene Tissue-treated specimens, do not use reagents contaminated with formalin. Residual formalin can lead to significant reduction of nucleic acid yield and integrity from PFPE tissue samples (see Technical Note "Influence of formalin contamination during processing of PAXgene Tissue fixed, paraffin-embedded tissue (PFPE) on RNA yield, integrity, and performance in quantitiative RT-PCR"). We recommend keeping alcohol for processing PAXgene Tissue-treated samples separate from alcohol used for processing formalin-fixed samples for at least the first five positions/steps in the processing. With this precaution, it is possible to process PAXgene Tissue-fixed and formalin-fixed samples on the same instrument.

4. Is a special processing protocol needed for the PAXgene Tissue System?
To prevent biomolecule degradation during processing, dehydration must start with at least 70–100% ethanol. We recommed using low-melting paraffin (melting point ≤56°C) and incubation in liquid paraffin for no longer than 3 hours.

Processing protocols for the PAXgene Tissue System are listed in the appendix of "PAXgene Tissue FIX Container (50 ml) Handbook".

5. Is it possible to integrate the PAXgene Tissue STABILIZER into automated tissue processing?
Yes. PAXgene Tissue STABILIZER can be used to fill the first position of a tissue processor. See the appendix of the "PAXgene Tissue FIX Container (50 ml) Handbook" for processing protocols with integrated PAXgene Tissue STABILIZER. When the STABILIZER is included as the first step of a protocol, tissues can be transferred from PAXgene Tissue FIX directly into the first processing position.

6. Is it possible to archive PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissue blocks?
Tissue morphology is preserved in PFPE tissue when stored at room temperature. However, biomolecules within paraffin blocks will undergo slow chemical degradation. For best preservation of morphology and maintenance of biomolecule integrity within the paraffin-embedded tissue, store PFPE blocks refrigerated at 5°C (2–8°C) or ideally frozen at –20°C (–15°C to –30°C). See poster "RT-PCR Performance of RNA Obtained from Archived FFPE and PFPE Blocks of Tissue" under Resources.

7. Is it possible to embed samples fixed and stabilized with the PAXgene Tissue System in Optimal Cutting Temperature (OCT) medium for freezing?
Yes. PreAnalytiX has developed a workflow and protocols for cryo-embedding tissue specimens fixed and stabilized in the PAXgene Tissue FIX Container (50 ml). Supplementary protocols for generating PAXgene Tissue-fixed, cryo-embedded (PFCE) tissues are available under Resources.

Compatibility with conventional pathology techniques

1. Is the morphology after H&E staining comparable to formalin-fixed samples?
Yes. Comparable morphology was observed in adjacent pieces from a tissue sample fixed either with neutral-buffered formalin or with the PAXgene Tissue System for a variety of human and animal tissues (Gündisch et al. 2014;Kap et al. 2011). Examples are provided in the Tissue Atlas. PAXgene Tissue treated-specimens have a tendency to be more eosinophilic. If an identical staining pattern to formalin-fixed samples is required, the incubation time in eosin should be reduced.

2. Are immunohistochemistry (IHC) assays developed for formalin-fixed, paraffin-embedded (FFPE) tissues compatible with PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissues?
Most antibodies used in IHC assays were developed for use with formalin-fixed tissue and include steps for unmasking epitopes. When working with PAXgene Tissue-treated specimens, test each antibody to determine if it is necessary to perform antigen-retrieval steps. In addition, it may be necessary to optimize antigen-retrieval steps or adjust antibody concentrations in PFPE tissue to achieve optimal staining intensities (see Technical Note "Effect of epitope retrieval conditions on immunohistochemical staining of PFPE tonsil tissue with anti-human Ki-67 antigen (clone MIB-1)" under Resources). Examples for IHC staining of adjacent human tissue sections fixed with neutral-buffered formalin or with PAXgene Tissue reagents are provided in the Tissue Atlas.

3. Can sections of PAXgene Tissue fixed, paraffin-embedded (PFPE) tissue be used for other histochemical staining techniques, such as PAS?
Human tissue samples treated with the PAXgene Tissue System were successfully used for periodic acid schiff (PAS), resorcin fuchsin, sirius red and Gomori staining (Kap et al. 2011). However, to achieve the same staining intensities with both PFPE and FFPE samples, it may be necessary to adjust incubation times.

4. Can PAXgene Tissue-fixed, paraffin-embedded tissue be used for in situ hybridization?
Yes, human tissue samples treated with the PAXgene Tissue System have been successfully used for fluorescence in situ hybridization (FISH). See the supplementary protocol "Preparation of PFPE tissue sections for use with in situ hybridization (ISH) staining" and Oberauner-Wappis et al. 2016 under Resources.

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 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. 2013. For examples of RNA integrity from clinical samples, see Viertler et al. 2012.

5. How well is DNA integrity preserved in PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissues?
In contrast to DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue, DNA from PFPE tissue exhibits high molecular weight. In most cases, a distinct 10 kb band is observed in electrophoretically separated DNA eluates. For an example, see Figure 2 in the Technical Note “Quantitative analysis of KRAS and BRAF mutational status in DNA from PAXgene Tissue fixed, paraffin-embedded (PFPE) tissue using Pyrosequencing technology“ under Resources.

6. 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 is of high quantity and quality, comparable to PFPE tissue.

7. 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, including miRNA, and DNA from PFCE tissue. Supplementary protocols developed specifically for the extraction of biomolecules from PFCE samples are available under the Resources tab.

8. Can proteins be extracted from PAXgene Tissue-fixed specimens?
Yes. Supplementary protocols are available for the purification of full-length proteins from PAXgene Tissue fixed (PF) tissue and paraffin blocks using the Qproteome FFPE Tissue Kit (QIAGEN, cat. no. 37623). For more information, see the corresponding supplementary protocols under the Resources tab.

9. 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 PFPE and PFCE tissue blocks for manual and laser microdissection are available under Resources.

10. 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 total RNA, including 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 the Resources tab.

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. 2013 and Figure 3 in Viertler et al. 2012.

2. What is the PCR performance of DNA purified from PAXgene Tissue-fixed, paraffin-embedded (PFPE) and PAXgene Tissue-fixed, cryo-embedded (PFCE) tissues compared to DNA from snap-frozen or formalin-fixed paraffin-embedded (FFPE) tissues?
In contrast to FFPE, DNA purified from PFPE and PFCE is of high molecular weight and free of chemical modifications. In demanding downstream applications, such as multiplex or long-range PCR, it performs similarly or identically to DNA isolated from frozen tissue. For examples, see, Figure 5 in Viertler et al. 2012.

3. Is DNA purified from PAXgene Tissue-fixed, paraffin-embedded (PFPE) tissues suitable for targeted NGS analysis?
Yes. DNA purified from PFPE is of high molecular weight and free of chemical modifications. Several independent researchers successfully used DNA from PFPE for NGS with different sample types, workflows and platforms (data in preparation). The data confirm recent suggestions that some DNA sequence artifacts associated with FFPE can be avoided with PFPE (Högnäs et al. 2017).

4. How is the quality of proteins purified from tissues fixed and stabilized with the PAXgene Tissue System?
Proteins from PAXgene Tissue-fixed, paraffin-embedded (PFPE) and PAXgene Tissue-fixed, cryo-embedded (PFCE) tissues are non-degraded, immunoreactive and have been successfully investigated by western blot analysis, reverse-phase protein arrays, two-dimensional gel electrophoresis (2D-PAGE), enzyme-linked immunosorbent assay (ELISA) and MALDI imaging mass spectrometry.

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