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PAXgene® Tissue System (RUO)Two worlds in one sample
PAXgene is a tradmark of PreAnalytiX GmbH. All other brands are trademarks of theire respective owners. © 2010-2016 PreAnalytiX.
01.05.2023
New workflows & applicationsDr. Tomasz Krenz
01.05.2023 3
PAXgene Tissue System* (RUO) Agenda
About PreAnalytiXPAXgene Tissue SystemWorkflow 1: PF-tissueWorkflow 2: PFPE-tissueWorkflow 3: PFCE-tissueLaser capture microdissectionReferences / Web resources
*The PAXgene Tissue applications presented here are 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
01.05.2023 4
About PreAnalytiX GmbHA QIAGEN/BD Joint Venture since 1998
BD• Specimen collection & stabilization
• Storage & transport
QIAGEN• Isolation of nucleic acids & other
biomolecules
• Automation
• Analysis
01.05.2023 5
PreAnalytiX GmbHOur Mission
Preanalytical errors cost ~ $460,000 / year in a typical German hospital Frost & Sullivan, 2011 survey on behalf of BD
Postanalytics
Preanalytics
68%
13%
19%
Analytics
“Preanalytical errors still account for nearly 60–70% of all problems occurring in laboratory diagnostics, … attributable to mishandling procedures during collection, handling, preparing or storing the specimens.” Lippi, G. et al. (2011) Preanalytical quality improvement: from dream to reality. Clin Chem Lab Med 49, 1113−1126. Epub 2011 Apr 25.
01.05.2023 6
PAXgene Tissue System* (RUO) Agenda
About PreAnalytiXPAXgene Tissue SystemWorkflow 1: PF-tissueWorkflow 2: PFPE-tissueWorkflow 3: PFCE-tissueLaser capture microdissectionReferences / Web resources
*The PAXgene Tissue applications presented here are 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
01.05.2023 7
Morphology Nucleic Acid Preservation Protein Preservation
PAXgene Tissue System √ √ √Competitor A
Morphology destroyed or compromised √ -
Competitor B Morphology destroyed or compromised
√ √
Formalin √ Crosslinking of biomolecules;Lack of standardization; no stabilization of biomolecules
PAXgene Tissue: Product PositioningSimultaneous preservation of morphology and biomolecules
PAXgene Tissue enables combined histological and multimodal analysis of biomolecules from the same sample
01.05.2023 8
PAXgene Tissue SystemSystem components
Collection, fixation and stabilizationPAXgene Tissue Container (10) containing PAXgene Tissue FIX and STABILIZER
dual chamber: 1 histocassette
PAXgene Tissue FIX Container (10 x 50 ml) + PAXgene Tissue STABILIZER concentrate
(8 x 150 ml for 4 liters of STABILIZER reagent each) single chamber: up to 4 histocassettes or larger tissue
sample
+
RNA RNA & miRNA DNA
Protein
Qproteome FFPE Tissue KitPAXgene Tissue Kits
Extraction
01.05.2023 9
PAXgene Tissue System* (RUO) Agenda
*The PAXgene Tissue applications presented here are 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
About PreAnalytiXPAXgene Tissue SystemWorkflow 1: PF-tissueWorkflow 2: PFPE-tissueWorkflow 3: PFCE-tissueLaser capture microdissectionReferences / Web resources
01.05.2023 10
AnalysisBiomolecule purification
Storage at −20 to −80°C
PAXgene Tissue: Workflows1. PF-Tissue
Tissue fixation, stabilization & transport
Tissue resection
Fixation2–48 hours (depending on tissue size)
Stabilization & storageUp to 7 days at RTUp to 4 weeks at 2–8°CUp to 5 years at –20°C (studies ongoing)Up to 6 years at –80°C (studies ongoing)
Biomolecule purificationPAXgene Tissue RNA kitPAXgene Tissue miRNA kitPAXgene Tissue DNA kitQproteome® FFPE kit & supplementary protocol
PF-Tissue
≤ 15 x 15 x 4 mm
PAXgene TissueContainer
PAXgene TissueFIX Container (50ml)
Cryo Vial
RNA, DNA, Protein
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1. PF-Tissue WorkflowHigh-quality NA from different tissue types including fibrous or fatty tissues
Φ RIN
- 21kb
RNA
DNA
01.05.2023 12
PAXgene Tissue System* (RUO) Agenda
*The PAXgene Tissue applications presented here are 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
About PreAnalytiXPAXgene Tissue SystemWorkflow 1: PF-tissueWorkflow 2: PFPE-tissueWorkflow 3: PFCE-tissueLaser capture microdissectionReferences / Web resources
01.05.2023 13
AnalysisBiomolecule purification& Staining
LaserCaptureMicrodisection
SectioningProcessing & embedding
Storage at -20 to -80°C
Tissue fixation, stabilization & transport
Paraffin-Embedding
PFPE-Tissue
RNA DNA Protein
H&E IHC ISH
PF-Tissue
PAXgene TissueContainer
PAXgene TissueFIX Container (50ml)
Microtome
Cryo Vial
LCM
Processing and paraffin embeddingPosition #1 of tissue processor: STABILIZER or ≥ 80% EtOHLow melting point paraffin (m.p. 54°C)Inkubation in paraffin < 3 hours
StainingHistochemical (H&E, PAS, …)ImmunohistochemicalIn situ hybridization
PAXgene Tissue: Workflows2. PFPE-Tissue
Biomolecule purificationPAXgene Tissue RNA kitPAXgene Tissue miRNA kitPAXgene Tissue DNA kitQproteome® FFPE kit & supplementary protocol
01.05.2023 14
2. PFPE-Tissue WorkflowPreservation of tissue morphology
Liver Spleen Kidney Intestine Lung Brain
H&E staining
01.05.2023 15
2. PFPE-Tissue WorkflowIHC & ISH with clinical samples
PFPE
FFPE
IHC of human tonsil:
Ki-67, clone MIB-1
ISH of human breast cancer: HER2 FISH
01.05.2023 16
2. PFPE-Tissue WorkflowEvaluation of RNA quality with clinical samples
Viertler et al. (2012) J Mol Diagn 14, 458.
RNA integrity from human liver:
PFPE vs. FFPE vs. snap frozen
Gene expression from human liver:qRT-PCR on a TaqMan array plate92 cancer pathway genes PFPE: Strong correlation (R² = 0.99)
to snap-frozen FFPE: large gene-to-gene variation (R² = 0.81)
01.05.2023 17
2. PFPE-Tissue WorkflowEvaluation of DNA quality with clinical samples
DNA integrity from human colorectal cancer:
PFPE: High molecular mass, suited for multiplex and long-range PCRFFPE: No recovery of large DNA molecules
Viertler et al. (2012) J Mol Diagn 14, 458.
01.05.2023 18
2. PFPE-Tissue WorkflowAnalytical protein techniques
Reverse-phase protein arrays
Western blot
Maldi-imaging mass spectrometry
2D-PAGE
ELISA (denatured proteins)
Ergin, B. et al. (2010) J Proteome Res 9, 5188−5196.
Ergi, B. et al. (2010)
Gündisch, S. et al.(2013) PLoS One 8, e60638.
Gündisch, S. et al. (2013)
Gündisch, S. et al. (2013)
01.05.2023 19
PAXgene-Tissue WorkflowAutomated purification of genomic DNA and RNA from PF & PFPE
QIAcube protocols available: significant savings of hands-on time
RNA from sections of PFPE tissueRNA from PF tissueDNA from section of PFPE tissueDNA from PF Tissue
01.05.2023 20
PAXgene Tissue System* (RUO) Agenda
*The PAXgene Tissue applications presented here are 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
About PreAnalytiXPAXgene Tissue SystemWorkflow 1: PF-tissueWorkflow 2: PFPE-tissueWorkflow 3: PFCE-tissueLaser capture microdissectionReferences / Web resources
01.05.2023 21
AnalysisBiomolecule purification& staining
LaserCaptureMicrodissection
SectioningProcessing & embedding
Storage at −20 to −80°C
PAXgene Tissue: New WorkflowsPFCE: PAXgene fixed, cryo-embedded
Tissue fixation, stabilization & transport
Cryo-Embedding
PFCE-Tissue
RNA DNA Protein
PF-Tissue
PAXgene TissueContainer
PAXgene TissueFIX Container (50ml)
Cryotome
Cryo Vial
LCM
Processing and cryo embeddingWash in 30% [w/v] sucrose at 2-8°COverlay Tissue with cryo-embedding media (e.g. Leica FCS22)Freeze in pre-chilled Isopentane or liquid N2
H&E IHC ISH
Biomolecule purification and Staining
Supplementary Protocols
01.05.2023 22
3. PFCE-Tissue WorkflowA. Preparation in isopentane on dry ice
< 2 x 15 x 15 mm
1. Tissue fixation and sucrose incubationTissue resection Tissue fixation &
stabilizationON incubation in 30% [w/v] sucrose
Freezing in isopentanecooled on dry ice
2. Snap-freezing in isopentane and embedding in cryo-media
Frozen tissue added to cryomold filled with cryo-embedding medium
Specimen disk placed on top
Cryomold submerged into isopentane
PFCE specimen
01.05.2023 23
< 2 x 15 x 15 mm
1. Tissue fixation and sucrose incubation Tissue resection Tissue fixation &
stabilizationON incubation in 30% [w/v] sucrose
2. Embedding in cryo-media and snap-freezing in liquid nitrogen
Tissue placed in cryomold filledwith cryo-embedding media
Specimen disk Placed on top
PFCE specimen
Snap freezing in liquid nitrogen
3. PFCE-Tissue WorkflowB. Preparation with liquid nitrogen
01.05.2023 24
3. PFCE-Tissue WorkflowKits & Protocols
Object PAXgene Tissue Supplementary Protocols Kits / ProductsWorkflow -Preparation of PFCE samples
Cryo-embedding tissue specimens fixed and stabilized with the PAXgene® Tissue System
PAXgene Tissue ContainerPAXgene Tissue FIX Container + Stabilizer
DNAPurification of genomic DNA from sections ofPAXgene® Tissue fixed, cryo-embedded (PFCE)tissue
PAXgene Tissue DNA Kit
RNAPurification of total RNA from sections ofPAXgene® Tissue fixed, cryo-embedded (PFCE)tissue
PAXgene Tissue RNA Kit
miRNAPurification of total RNA, including miRNA, from sections of PAXgene® Tissue fixed, cryo-embedded (PFCE) tissue
PAXgene Tissue miRNA Kit
ProteinPurification of full-length proteins from sections of PAXgene® Tissue fixed, cryo-embedded (PFCE) tissue
Qproteome FFPE Kit
25Date Text, Location
Liver Spleen
Heart muscleIntestine
3. PFCE-Tissue WorkflowHistomorphology
H&E staining
01.05.2023 26
3. PFCE-Tissue WorkflowHigh-quality NA from different tissue types
RIN: 8.5 RIN: 7.6RIN: 8.9 RIN: 9.1RIN: 8.6
RNA
- 21kb
DNA
Liver Spleen Intestine
Heart muscle
Lung
01.05.2023 27
Data kindly provided by Karl-Friedrich Becker, Technical University of Munich, Germany
Liver StomachSection of PFCE tissue
mounted on slide
Cryo-medium removed
Area of interest removed manually or by laser microdissection
Collected into Qproteome FFPE buffer EXB Plus
Incubated for 10 min @95°C
3. PFCE-Tissue WorkflowFull-length (phospho-)proteins from PFCE tissue
SDS-PAGE & western blot
12.5 µg protein per lane
01.05.2023 28
PAXgene Tissue System* (RUO) Agenda
*The PAXgene Tissue applications presented here are 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
About PreAnalytiXPAXgene Tissue SystemWorkflow 1: PF-tissueWorkflow 2: PFPE-tissueWorkflow 3: PFCE-tissueLaser capture microdissectionReferences / Web resources
01.05.2023 29
BiomoleculePurificationLaser Capture MicrodissectionStaining (H&E)
Sectioning/ removal of embedding medium
Processing & embedding
Paraffin-Embedding
Cryo-Embedding
PFPE-Tissue
PFCE-Tissue
Microtome
Cryotome
RNA DNALMD (e.g. Leica 6500)H&E
PFPE workflow
PFCE workflow
PAXgene Tissue: New WorkflowsLaser Capture Microdissection (LCM)
01.05.2023 30
MicrodissectionSet Up
4. Laser Capture Microdissection (LCM)Procedure
Sample Processing
Slidepreparation
PFPE/PFCE tissue sections Microscope + Sample
LCM Software
Collection tubesstain
Definition of region
Cutting
Collection
Collection in Lysis buffer
Purification of genomic DNA Purification of total RNAPurification of total RNA, including miRNA
P1000877.MP4
01.05.2023 31
Object PAXgene Tissue Supplementary Protocols Kits / Products
Workflow -Preparation of slides
Preparation of sections from PAXgene® Tissue fixed, paraffin-embedded (PFPE) and PAXgene Tissue fixed, cryo-embedded (PFCE) tissues for manual or laser microdissection (LMD)
PAXgene Tissue ContainerPAXgene Tissue FIX Container + Stabilizer
DNAPurification of genomic DNA from microdissected PAXgene® Tissue-fixed, paraffin-embedded (PFPE) and PAXgene Tissue-fixed, cryo-embedded (PFCE) tissues
PAXgene Tissue DNA Kit
RNAPurification of total RNA from microdissected PAXgene® Tissue fixed, paraffin-embedded (PFPE) and PAXgene Tissue fixed, cryo-embedded (PFCE) tissues
PAXgene Tissue RNA Kit
miRNAPurification of total RNA, including miRNA, from microdissected PAXgene® Tissue fixed, paraffin embedded (PFPE) and PAXgene Tissue fixed, cryoembedded (PFCE) tissues
PAXgene Tissue miRNA Kit
4. Laser Capture Microdissection (LCM)Kits & Protocols
01.05.2023 32
liver
sple
en
colo
n
liver
sple
en
colo
n
liver
sple
en
colo
n
PFPE PFCE FFPE[nt] High-quality and high-molecular−weight RNA Agilent Bioanalyzer: Gel and corresponding RIN values of total RNA from an area of 1 mm² (4,000−12,000 cells) ß-act RT PCR
4. Laser Capture Microdissection (LCM)RNA from PFPE & PFCE tissue
# cells
01.05.2023 33
PFPE PFCEliv
er
sple
en
colo
n
liver
sple
en
colo
n
High-quality miRNA Agilent Bioanalyzer Small RNA: Gel with calculated miRNA content and ElectropherogramsArea of 1 mm² (4,000−12,000 cells) selected
[nt]
4. Laser Capture Microdissection (LCM)miRNA from PFPE & PFCE tissue
01.05.2023 34
Total yield and performance (ß-act PCR)Area of 0.005−1 mm² (20−12,000 cells) selected
4. Laser Capture Microdissection (LCM)DNA from PFPE & PFCE tissue
01.05.2023 35
PAXgene Tissue System* (RUO) Agenda
*The PAXgene Tissue applications presented here are 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
About PreAnalytiXPAXgene Tissue SystemWorkflow 1: PF-tissueWorkflow 2: PFPE-tissueWorkflow 3: PFCE-tissueLaser capture microdissectionReferences / Web resources
01.05.2023 36
ReferencesProjects / Collaborations
SPIDIA
STRATFix UK
Genotype Tissue Expression Project (GTEx)
CBmed Austria
Barret Syndrome Project
Genesis
Enabling stratified medicine with novel fixatives for improved pre-analytical pathology workflows
Next generation sequencing in molecular pathology diagnostics
Identification of Biomarkers for the Development of Adenocarcinoma from Barrett-Esophagus
Genetic Biopsy for Prediction of Surveilance Intervals after Endoscopic Resection of Colonic Polyps
Standardization and Improvement of Generic Pre-analytical Tools and Procedures for In Vitro Diagnostics
7 research organizations, 8 companies, 1 standards organization (CEN), Coordinated by QIAGEN
8 clinical sides, 1 company, Coordinated by QIAGEN
Establish a resource database and tissue bank to study the relationship between genetic variation and gene expression
Over 700 donors, >21,000 collected tissue samples
Up to 2000 patients
220 samples, 100 patients; follow up project with ~10.000 patients
01.05.2023 37
ReferencesScientific articles in peer-reviewed journals
Author Titel Journal ContentYamaguchi, et al. Comprehensive DNA Methylation and Extensive Mutation Analyses of HER2-Positive Breast
Cancer.Oncology. 2015 Jan; [Epub ahead of print]
409 cancer-related genes/ bench-top NGS/ DNA methylation statuses/ bead array with 485,512 probes
Keen J and Moore H The Genotype-Tissue Expression (GTEx) project: Linking Clinical Data with Molecular Analysis to Advance Personalized Medicine.
J.Pers. Med. 2015 Feb;5:22-29
Whole genome and RNA sequencing
Andersen GB, et al. Improved reproducibility in genome-wide DNA methylation analysis for PAXgene® fixed samples compared to restored FFPE DNA.
Anal Biochem. 2014 Sep; 468C:50-58. Genome-wide DNA methylation analysis/ Illumina 450K BeadChip
Gündisch S, et al. Evaluation of colon cancer histomorphology: a comparison between formalin and PAXgene tissue fixation by an international ring trial.
Virchows Arch. 2014 Nov; 465(5):509-19.
Blinded, randomized ring trial by 20 pathologists; virtual microscopy; histological grading and quality parameter
Kap M, et al. Inactivation of Influenza A virus, Adenovirus, and Cytomegalovirus with PAXgene Tissue Fixative and Formalin.
Biopreservation and Biobanking. 2013 Aug; 11(4): 229-234
Viral inactivation characteristics by PAXgene Tissue fixative
Staff S, et al. Preservation of nucleic acids and tissue morphology in paraffin-embedded clinical samples: comparison of five molecular fixatives.
J Clin Pathol. 2013 Sep;66(9):807-10
Comparison study Z7, RCL2, PAXgene Tissue, Allprotect, RNAlater, Formlin; DNA, RNA, Morphology, IHC, FISH, CISH
Oetjen J, et al. MRI-compatible pipeline for three-dimensional MALDI imaging mass spectrometry using PAXgene fixation.
J Proteomics. 2013 Sep;90:52-60
Computational pipeline for molecular analysis combining 3D MALDI imaging, magnetic resonance imaging and H&E
Lonsdale, et al. The Genotype-Tissue Expression (GTEx) project. Nature Genetics 2013 Jun; 45, 580-585
Description of GTEx project
Gündisch S, et al. The PAXgene Tissue System Preserves Phosphoproteins in Human Tissue Specimens and Enables Comprehensive Protein Biomarker Research.
PLoS One. 2013;8(3):e60638
Proteome including phosphoproteome analysis; fresh frozen, PFPE, FFPE, western blotting, 2D-PAGE, ELISA
Belloni B, et al. Will PAXgene substitute formalin? A morphological and molecular comparative study using a new fixative system.
J Clin Pathol. 2013 Feb;66(2):124-35
Melanoma fresh frozen, PFPE, FFPE, morphology, IHC, DNA, RNA
Groelz D, et al. Non- Formalin Fixative versus Formalin-fixed Tissue: A Comparison of Histology and RNA Quality.
Exp Mol Pathol. 2013 Feb;94(1):188-94
Morphology and RNA preservation; comparison PFPE and FFPE; RT-qPCR
Viertler C, et al. A New Technology for Stabilization of Biomolecules in Tissues for Combined Histological and Molecular Analyses.
J Mol Diagn. 2012 Sep;14(5):458-66
RNA, DNA, miRNA preservation, fresh frozen, PFPE, FFPE, expression profiling
Trede D, et al. Exploring three-dimensional matrix-assisted laser desorption/ionization imaging mass spectrometry data: three-dimensional spatial segmentation of mouse kidney.
Anal Chem. 2012 Jul;84(14):6079-87.
3D MALDI imaging, 3D spatial segmentation
Baker M Biorepositories: Building better biobanks. Nature 2012 Jun;486(7401):141-6.
Efforts to improve biobanking
Kap M, et al. Histological Assessment of PAXgene Tissue Fixation and StabilizationReagents.
PLoS ONE. 2011;6(11)
Quality assessment PFPE vs FFPE, 20 different human tissue types, H&E, IHC, 4 pathologists, histological grading and quality
Ergin B, et al. Proteomic analysis of PAXgene-fixed tissues. J Proteome Res. 2010 Oct;9(10):5188-96
Proteome analysis from fresh frozen, PFPE and FFPE; western blotting, reverse-phase microarrays, MALDI imaging MS
Loibner et al. (2016) Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative.
PLoS One. 2016 Mar 14;11(3)
Oberauner-Wappis et al. (2016)
Protocol for HER2 FISH determination on PAXgene-fixed and paraffin-embedded tissue in breast cancer
Accepted in International Journal of Experimental Pathology
Carithers et al. (2015)
A novel approach to high-quality postmortem tissue procurement: the GTEx project
Biopreserv Biobank. 2015 Oct;13(5):311-9.
Hara et al. (2015) Surgical Specimens of Colorectal Cancer Fixed with PAXgene Tissue System Preserve High-Quality RNA
Biopreserv Biobank. 2015 Oct;13(5):325-34.
Gillard, M. et al. (2015)
Next-gen tissue: preservation of molecular andmorphological fidelity in prostate tissue.
Am J Transl Res 7:1227-1235
Yamaguchi, T. et al. (2015)
Comprehensive DNA Methylation and Extensive Mutation Analyses of HER2-Positive Breast Cancer.
Oncology. 2015;88(6):377-84
Loibner et al. (2016) Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative.
PLoS One. 2016 Mar 14;11(3)
Oberauner-Wappis et al. (2016)
Protocol for HER2 FISH determination on PAXgene-fixed and paraffin-embedded tissue in breast cancer
Accepted in International Journal of Experimental Pathology
Carithers et al. (2015)
A novel approach to high-quality postmortem tissue procurement: the GTEx project
Biopreserv Biobank. 2015 Oct;13(5):311-9.
Hara et al. (2015) Surgical Specimens of Colorectal Cancer Fixed with PAXgene Tissue System Preserve High-Quality RNA
Biopreserv Biobank. 2015 Oct;13(5):325-34.
Gillard, M. et al. (2015)
Next-gen tissue: preservation of molecular andmorphological fidelity in prostate tissue.
Am J Transl Res 7:1227-1235
Yamaguchi, T. et al. (2015)
Comprehensive DNA Methylation and Extensive Mutation Analyses of HER2-Positive Breast Cancer.
Oncology. 2015;88(6):377-84
01.05.2023 38
Web Resources Technical Support
Technical notesPI3K mutation analysisIHC assay optimization (Ki67)Formalin contamination during processingVacuum sealing for fixed and stabilized tissues for transportRNA yield, purity and integrity from PFPE rat tissueKRAS and BRAF mutational status using Pyrosequencing technology…
Supplementary protocolsManual processingDeparaffinization with Deparaffinization SolutionPurification of proteins from sections of PFPE tissueAllprep: Simultaneous purification of RNA and DNA from sections of PFPE tissuePurification of RNA/DNA from microdissected PFPE and PFCE tissue
IHC application list
Manual stainingBenchmark XTMicrowaveLeica Bond MaxDako Autostainer
OtherHandbooksPostersBrochuresList of references in peer reviewed journalsFAQ ListSafety data sheets
Constantly growing list of technical support: http://www.preanalytix.com/products/tissue/fixation-stabilization/paxgene-tissue-container
01.05.2023 39
Web ResourcesTissue Atlas
http://www.preanalytix.com/products/tissue/tissue-atlasHuman large intestine Rat retina Rat tongue
Rat kidney Human large intestine Human stomach
H&E
PAS
IHC
Human tonsil Human colorectal cancer Human breast cancer Human tonsil
Human liver
Rat skin sebaceous glands
40
Q&A session
01.05.2023
Thank you for your attention!
For up-to-date licensing information and product-specific disclaimers for QIAGEN products, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or www.preanalytix.com or can be requested from QIAGEN Technical Services or your local distributor.
41
Q&A session
01.05.2023
Supplement
For up-to-date licensing information and product-specific disclaimers for QIAGEN products, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or www.preanalytix.com or can be requested from QIAGEN Technical Services or your local distributor.
01.05.2023 42
1. PF-Tissue WorkflowFull-length (phospho-)proteins from PF tissue
Data kindly provided by Karl-Friedrich Becker, Technical University of Munich, Germany
12.5 µg protein per lane
Up to 100 mg of PF tissue washed with PBS
Disrupted and homogenized with TissueLyser
Incubated for 10 min @95°C
Collected into Qproteome FFPE buffer EXB Plus
Liver Stomach
SDS-PAGE & western blot
01.05.2023 43
ReferencesSPIDIA
Main FactsFor “Standardization and Improvement of Generic Pre-analytical Tools and Procedures for In Vitro Diagnostics”Funded by FP7-HEALTH (EC)7 public research organizations, 8 companies, 1 standards organization (CEN)Coordinated by QIAGENRun time from Oct. 2008 – March 2013
Main GoalsNew preanalytical tools & technologies (blood, plasma, tissue, swabs)Sample quality markers (blood, tissue)Pan-European guidelines for preanalytics (blood, tissue) Training and dissemination
01.05.2023 44
ReferencesThe Genotype Tissue Expression Project (GTEx)
NIH common fund initiativeAim: establish a resource database and tissue bank to study the relationship between genetic variation and gene expressionOver 700 donors (>21,000 tissue samples) have been collected to datePFPE-tissue for histological examinationRNA from PF-tissue for RNA sequencing
”PAXgene is at least as good as formalin for histology. It is now our only morphologic fixative.” OBBR pathologists Philip Branton, John Madden James Robb, Leslie Sobin, Carolyn Comptonhttp://www.genome.gov/Pages/About/NACHGR/September2011AgendaDocs/NACHGR_Sep122011_%20GTExUpdate_Struewing.pdf
“Each tissue is preserved in PAXgene® tissue fixative …sections are examined by a team of board certified pathologists …images and histological data will be available to researchers …PAXgene® tissue-fixed samples are then shipped to … for whole genome and RNA sequencing.”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, 22−29.
01.05.2023 45
ReferencesThe Barret Syndrome Project
endoscopic tissue retrieval
PAXgene Tissue fixation & stabilization,transport and processing
H&E, PAS – staining for diagnosisIHC stainingDNA, RNA purification
Whole genome amplificationNext-generation sequencing
m4 Cluster Munich fund initiative — collaboration with Technical University MunichIdentification and Analysis of Biomarkers for the Development of Adenocarcinoma from Barrett-EsophagusAims: Optimization of pathogenesis, diagnosis and therapy of esophageal cancer
Identification of predictive biomarkers in Barret syndrome Implementation of PAXgene Tissue for clinical application Validation of tumor evolution theory (dysplasia caused by cardia stem cells) Up to 2000 patients
01.05.2023 46
ReferencesThe STRATFix Project
“Enabling stratified medicine with novel fixatives for improved preanalytical pathology workflows”Formaldehyde free workflows in clinical routineSolid tissue, fine needle biopsies, bloodPartners: