Quantitative Gene Expression Analysis in Microdissected Archival Formalin-Fixed and Paraffin-Embedded Tumor Tissue
Formalin-fixed, paraffin-embedded tissue is the most widely available material for retrospective clinical studies. In combination with the potential of genomics, these tissues represent an invaluable resource for the elucidation of disease mechanisms and validation of differentially expressed genes...
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| Veröffentlicht in: | The American journal of pathology 2001-02, Vol.158 (2), p.419-429 |
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| creator | Specht, Katja Richter, Thomas Müller, Ulrike Walch, Axel Werner, Martin Höfler, Heinz |
| description | Formalin-fixed, paraffin-embedded tissue is the most widely available material for retrospective clinical studies. In combination with the potential of genomics, these tissues represent an invaluable resource for the elucidation of disease mechanisms and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators. We describe here an approach that, in combination with laser-assisted microdissection allows quantitative gene expression analysis in formalin-fixed, paraffin-embedded archival tissue. Using an optimized RNA microscale extraction procedure in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction based on fluorogenic TaqMan methodology, we analyzed the expression of a panel of cancer-relevant genes,
EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, and
HPRT and
PGK as controls. We demonstrate that expression level determinations from formalin-fixed, paraffin-embedded tissues are accurate and reproducible. Measurements were comparable to those obtained with matching fresh-frozen tissue and neither fixation grade nor time significantly affected the results. Laser microdissection studies with 5-μm thick sections and defined numbers of tumor cells demonstrated that reproducible quantitation of specific mRNAs can be achieved with only 50 cells. We applied our approach to HER-2/neu quantitative gene expression analysis in 54 microdissected tumor and nonneoplastic archival samples from patients with Barrett’s esophageal adenocarcinoma and showed that the results matched those obtained in parallel by fluorescence
in situ hybridization and immunohistochemistry. Thus, the combination of laser-assisted microdissection and real-time TaqMan reverse transcriptase-polymerase chain reaction opens new avenues for the investigation and clinical validation of gene expression changes in archival tissue specimens. |
| doi_str_mv | 10.1016/S0002-9440(10)63985-5 |
| format | Article |
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EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, and
HPRT and
PGK as controls. We demonstrate that expression level determinations from formalin-fixed, paraffin-embedded tissues are accurate and reproducible. Measurements were comparable to those obtained with matching fresh-frozen tissue and neither fixation grade nor time significantly affected the results. Laser microdissection studies with 5-μm thick sections and defined numbers of tumor cells demonstrated that reproducible quantitation of specific mRNAs can be achieved with only 50 cells. We applied our approach to HER-2/neu quantitative gene expression analysis in 54 microdissected tumor and nonneoplastic archival samples from patients with Barrett’s esophageal adenocarcinoma and showed that the results matched those obtained in parallel by fluorescence
in situ hybridization and immunohistochemistry. Thus, the combination of laser-assisted microdissection and real-time TaqMan reverse transcriptase-polymerase chain reaction opens new avenues for the investigation and clinical validation of gene expression changes in archival tissue specimens.</description><identifier>ISSN: 0002-9440</identifier><identifier>EISSN: 1525-2191</identifier><identifier>DOI: 10.1016/S0002-9440(10)63985-5</identifier><identifier>PMID: 11159180</identifier><identifier>CODEN: AJPAA4</identifier><language>eng</language><publisher>Bethesda, MD: Elsevier Inc</publisher><subject>Animals ; Biological and medical sciences ; Dissection ; Fixatives ; Formaldehyde ; Frozen Sections ; Gene Expression Regulation, Neoplastic ; HT29 Cells ; Humans ; Investigative techniques, diagnostic techniques (general aspects) ; Laser Therapy ; Medical sciences ; Mice ; Mice, Nude ; Microsurgery ; Miscellaneous. Technology ; Neoplasms - genetics ; Neoplasms - pathology ; Neoplasms, Experimental - genetics ; Neoplasms, Experimental - pathology ; Paraffin Embedding ; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques ; Receptor, ErbB-2 - genetics ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger - analysis ; RNA, Messenger - genetics ; RNA, Messenger - metabolism ; RNA, Neoplasm - analysis ; RNA, Neoplasm - genetics ; RNA, Neoplasm - metabolism ; Technical Advance ; Time Factors ; Tissue Fixation ; Tumor Cells, Cultured</subject><ispartof>The American journal of pathology, 2001-02, Vol.158 (2), p.419-429</ispartof><rights>2001 American Society for Investigative Pathology</rights><rights>2001 INIST-CNRS</rights><rights>Copyright American Society for Investigative Pathology Feb 2001</rights><rights>Copyright © 2001, American Society for Investigative Pathology</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c615t-ef5d3ce5948ab47d0487c2ba6bc0833f913ad474faea2d910d7d710b6c821f753</citedby><cites>FETCH-LOGICAL-c615t-ef5d3ce5948ab47d0487c2ba6bc0833f913ad474faea2d910d7d710b6c821f753</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1850313/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://dx.doi.org/10.1016/S0002-9440(10)63985-5$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,3548,27923,27924,45994,53790,53792</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=886683$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11159180$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Specht, Katja</creatorcontrib><creatorcontrib>Richter, Thomas</creatorcontrib><creatorcontrib>Müller, Ulrike</creatorcontrib><creatorcontrib>Walch, Axel</creatorcontrib><creatorcontrib>Werner, Martin</creatorcontrib><creatorcontrib>Höfler, Heinz</creatorcontrib><title>Quantitative Gene Expression Analysis in Microdissected Archival Formalin-Fixed and Paraffin-Embedded Tumor Tissue</title><title>The American journal of pathology</title><addtitle>Am J Pathol</addtitle><description>Formalin-fixed, paraffin-embedded tissue is the most widely available material for retrospective clinical studies. In combination with the potential of genomics, these tissues represent an invaluable resource for the elucidation of disease mechanisms and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators. We describe here an approach that, in combination with laser-assisted microdissection allows quantitative gene expression analysis in formalin-fixed, paraffin-embedded archival tissue. Using an optimized RNA microscale extraction procedure in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction based on fluorogenic TaqMan methodology, we analyzed the expression of a panel of cancer-relevant genes,
EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, and
HPRT and
PGK as controls. We demonstrate that expression level determinations from formalin-fixed, paraffin-embedded tissues are accurate and reproducible. Measurements were comparable to those obtained with matching fresh-frozen tissue and neither fixation grade nor time significantly affected the results. Laser microdissection studies with 5-μm thick sections and defined numbers of tumor cells demonstrated that reproducible quantitation of specific mRNAs can be achieved with only 50 cells. We applied our approach to HER-2/neu quantitative gene expression analysis in 54 microdissected tumor and nonneoplastic archival samples from patients with Barrett’s esophageal adenocarcinoma and showed that the results matched those obtained in parallel by fluorescence
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In combination with the potential of genomics, these tissues represent an invaluable resource for the elucidation of disease mechanisms and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators. We describe here an approach that, in combination with laser-assisted microdissection allows quantitative gene expression analysis in formalin-fixed, paraffin-embedded archival tissue. Using an optimized RNA microscale extraction procedure in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction based on fluorogenic TaqMan methodology, we analyzed the expression of a panel of cancer-relevant genes,
EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, and
HPRT and
PGK as controls. We demonstrate that expression level determinations from formalin-fixed, paraffin-embedded tissues are accurate and reproducible. Measurements were comparable to those obtained with matching fresh-frozen tissue and neither fixation grade nor time significantly affected the results. Laser microdissection studies with 5-μm thick sections and defined numbers of tumor cells demonstrated that reproducible quantitation of specific mRNAs can be achieved with only 50 cells. We applied our approach to HER-2/neu quantitative gene expression analysis in 54 microdissected tumor and nonneoplastic archival samples from patients with Barrett’s esophageal adenocarcinoma and showed that the results matched those obtained in parallel by fluorescence
in situ hybridization and immunohistochemistry. Thus, the combination of laser-assisted microdissection and real-time TaqMan reverse transcriptase-polymerase chain reaction opens new avenues for the investigation and clinical validation of gene expression changes in archival tissue specimens.</abstract><cop>Bethesda, MD</cop><pub>Elsevier Inc</pub><pmid>11159180</pmid><doi>10.1016/S0002-9440(10)63985-5</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | The American journal of pathology, 2001-02, Vol.158 (2), p.419-429 |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; ScienceDirect Journals (5 years ago - present); PubMed Central |
| subjects | Animals Biological and medical sciences Dissection Fixatives Formaldehyde Frozen Sections Gene Expression Regulation, Neoplastic HT29 Cells Humans Investigative techniques, diagnostic techniques (general aspects) Laser Therapy Medical sciences Mice Mice, Nude Microsurgery Miscellaneous. Technology Neoplasms - genetics Neoplasms - pathology Neoplasms, Experimental - genetics Neoplasms, Experimental - pathology Paraffin Embedding Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques Receptor, ErbB-2 - genetics Reverse Transcriptase Polymerase Chain Reaction RNA, Messenger - analysis RNA, Messenger - genetics RNA, Messenger - metabolism RNA, Neoplasm - analysis RNA, Neoplasm - genetics RNA, Neoplasm - metabolism Technical Advance Time Factors Tissue Fixation Tumor Cells, Cultured |
| title | Quantitative Gene Expression Analysis in Microdissected Archival Formalin-Fixed and Paraffin-Embedded Tumor Tissue |
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