Gene Expression Analysis in Microdissected Samples from Decalcified Tissues
OBJECTIVE:The aim of this study was to determine the impact of standard methods for processing decalcified highly mineralized tissues on RNA yield and quality from microdissected samples. DESIGN:Rat mandibles were fixed with either formalin-based or ethanol-based fixatives, decalcified in 20% ethyle...
Gespeichert in:
| Veröffentlicht in: | Diagnostic molecular pathology 2012-06, Vol.21 (2), p.120-126 |
|---|---|
| Hauptverfasser: | , , , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 126 |
|---|---|
| container_issue | 2 |
| container_start_page | 120 |
| container_title | Diagnostic molecular pathology |
| container_volume | 21 |
| creator | Salmon, Cristiane Ribeiro Silvério, Karina Gonzales Giorgetti, Ana Paula de Oliveira Sallum, Enilson Antonio Casati, Márcio Zaffalon Nociti, Francisco Humberto |
| description | OBJECTIVE:The aim of this study was to determine the impact of standard methods for processing decalcified highly mineralized tissues on RNA yield and quality from microdissected samples.
DESIGN:Rat mandibles were fixed with either formalin-based or ethanol-based fixatives, decalcified in 20% ethylenediaminetetraacetic acid solution for 15 days, and embedded in paraffin. Transversal sections of the molars were mounted on membrane glass slides for laser capture microdissection. Unfixed frozen liver samples were used as controls to determine the impact of fixatives, decalcification and paraffin embedding on RNA integrity and recovery after sample preparation, and laser microdissection. Total RNA was obtained from periodontal ligament and fresh-frozen liver; RNA quality was assessed by Bioanalyzer, and 5 ng of total RNA was used for cDNA synthesis followed by gene expression analyses by polymerase chain reaction using 3 sets of primers for glyceraldehyde 3-phosphate dehydrogenase.
RESULTS:Data analysis demonstrated that all fixed samples presented some level of RNA fragmentation as compared with fresh-frozen samples (P |
| doi_str_mv | 10.1097/PDM.0b013e31823e9395 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_cross</sourceid><recordid>TN_cdi_proquest_miscellaneous_1291615429</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><sourcerecordid>1013919976</sourcerecordid><originalsourceid>FETCH-LOGICAL-c3895-43dd056ca26b44b0629e1a3c475cb60d1d4e45a47227be7ff4cc1fb079da4f493</originalsourceid><addsrcrecordid>eNqNkE1PAjEQhhujEUT_gTF79LLYz93tkQCiEaKJeN50u7Oh2v2whSD_3hrQgwfjaSaZ553JPAhdEjwkWKY3T5PFEBeYMGAkowwkk-II9YlgLOY4SY5DjwWNpRCkh868f8UYM56JU9SjVAiBpeijhxk0EE0_Ogfem7aJRo2yO298ZJpoYbRrS-M96DWU0bOqOws-qlxbRxPQympTmTBYBmQD_hydVMp6uDjUAXq5nS7Hd_H8cXY_Hs1jzTIpYs7KEotEK5oUnBc4oRKIYpqnQhcJLknJgQvFU0rTAtKq4lqTqsCpLBWvuGQDdL3f27n2Pdxd57XxGqxVDbQbnxMqSUIEp_9Agz9JpEyTgPI9Gn723kGVd87Uyu0ClH8Zz4Px_LfxELs6XNgUNZQ_oW_FAcj2wLa1a3D-zW624PIVKLte_b37E_ZCjlY</addsrcrecordid><sourcetype>Aggregation Database</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1013919976</pqid></control><display><type>article</type><title>Gene Expression Analysis in Microdissected Samples from Decalcified Tissues</title><source>Journals@Ovid Ovid Autoload</source><source>MEDLINE</source><creator>Salmon, Cristiane Ribeiro ; Silvério, Karina Gonzales ; Giorgetti, Ana Paula de Oliveira ; Sallum, Enilson Antonio ; Casati, Márcio Zaffalon ; Nociti, Francisco Humberto</creator><creatorcontrib>Salmon, Cristiane Ribeiro ; Silvério, Karina Gonzales ; Giorgetti, Ana Paula de Oliveira ; Sallum, Enilson Antonio ; Casati, Márcio Zaffalon ; Nociti, Francisco Humberto</creatorcontrib><description>OBJECTIVE:The aim of this study was to determine the impact of standard methods for processing decalcified highly mineralized tissues on RNA yield and quality from microdissected samples.
DESIGN:Rat mandibles were fixed with either formalin-based or ethanol-based fixatives, decalcified in 20% ethylenediaminetetraacetic acid solution for 15 days, and embedded in paraffin. Transversal sections of the molars were mounted on membrane glass slides for laser capture microdissection. Unfixed frozen liver samples were used as controls to determine the impact of fixatives, decalcification and paraffin embedding on RNA integrity and recovery after sample preparation, and laser microdissection. Total RNA was obtained from periodontal ligament and fresh-frozen liver; RNA quality was assessed by Bioanalyzer, and 5 ng of total RNA was used for cDNA synthesis followed by gene expression analyses by polymerase chain reaction using 3 sets of primers for glyceraldehyde 3-phosphate dehydrogenase.
RESULTS:Data analysis demonstrated that all fixed samples presented some level of RNA fragmentation as compared with fresh-frozen samples (P<0.05). Samples fixed with Protocol (10% formalin) showed the least RNA fragmentation as compared with other fixatives (P<0.05), and biologically useful RNA was extracted even from microdissected samples with a minimum RNA Integrity Number of 1.5. Moreover, RNA fragments up to 396 bp were assayable by reverse transcriptase-polymerase chain reaction, although short-targeted fragments as 74 bp were more consistently amplified.
CONCLUSIONS:Although variable levels of RNA fragmentation should be expected, gene expression analysis can be performed from decalcified paraffin-embedded microdissected samples, with the best results obtained for short-targeted fragments around 70 bp.</description><identifier>ISSN: 1052-9551</identifier><identifier>EISSN: 1533-4066</identifier><identifier>DOI: 10.1097/PDM.0b013e31823e9395</identifier><identifier>PMID: 22555095</identifier><language>eng</language><publisher>United States: Lippincott Williams & Wilkins, Inc</publisher><subject>Animals ; Bone Demineralization Technique ; Chelating Agents - chemistry ; Data processing ; Decalcification ; Edetic Acid - chemistry ; Embedding ; Fixatives ; Formaldehyde ; Gene expression ; Gene Expression Profiling ; Glyceraldehyde-3-phosphate dehydrogenase ; Glyceraldehyde-3-Phosphate Dehydrogenases - genetics ; Glyceraldehyde-3-Phosphate Dehydrogenases - metabolism ; Laser Capture Microdissection ; Lasers ; Liver ; Liver - chemistry ; Liver - metabolism ; Mandible - chemistry ; Mandible - metabolism ; Mandibles ; Molar - chemistry ; Molar - metabolism ; Molars ; Paraffin ; periodontal ligament ; Periodontal Ligament - chemistry ; Periodontal Ligament - metabolism ; Polymerase chain reaction ; Primers ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; RNA ; RNA Stability ; RNA, Messenger - chemistry ; RNA, Messenger - genetics ; RNA, Messenger - isolation & purification ; Teeth ; Tissue Fixation</subject><ispartof>Diagnostic molecular pathology, 2012-06, Vol.21 (2), p.120-126</ispartof><rights>2012 Lippincott Williams & Wilkins, Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3895-43dd056ca26b44b0629e1a3c475cb60d1d4e45a47227be7ff4cc1fb079da4f493</citedby><cites>FETCH-LOGICAL-c3895-43dd056ca26b44b0629e1a3c475cb60d1d4e45a47227be7ff4cc1fb079da4f493</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22555095$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Salmon, Cristiane Ribeiro</creatorcontrib><creatorcontrib>Silvério, Karina Gonzales</creatorcontrib><creatorcontrib>Giorgetti, Ana Paula de Oliveira</creatorcontrib><creatorcontrib>Sallum, Enilson Antonio</creatorcontrib><creatorcontrib>Casati, Márcio Zaffalon</creatorcontrib><creatorcontrib>Nociti, Francisco Humberto</creatorcontrib><title>Gene Expression Analysis in Microdissected Samples from Decalcified Tissues</title><title>Diagnostic molecular pathology</title><addtitle>Diagn Mol Pathol</addtitle><description>OBJECTIVE:The aim of this study was to determine the impact of standard methods for processing decalcified highly mineralized tissues on RNA yield and quality from microdissected samples.
DESIGN:Rat mandibles were fixed with either formalin-based or ethanol-based fixatives, decalcified in 20% ethylenediaminetetraacetic acid solution for 15 days, and embedded in paraffin. Transversal sections of the molars were mounted on membrane glass slides for laser capture microdissection. Unfixed frozen liver samples were used as controls to determine the impact of fixatives, decalcification and paraffin embedding on RNA integrity and recovery after sample preparation, and laser microdissection. Total RNA was obtained from periodontal ligament and fresh-frozen liver; RNA quality was assessed by Bioanalyzer, and 5 ng of total RNA was used for cDNA synthesis followed by gene expression analyses by polymerase chain reaction using 3 sets of primers for glyceraldehyde 3-phosphate dehydrogenase.
RESULTS:Data analysis demonstrated that all fixed samples presented some level of RNA fragmentation as compared with fresh-frozen samples (P<0.05). Samples fixed with Protocol (10% formalin) showed the least RNA fragmentation as compared with other fixatives (P<0.05), and biologically useful RNA was extracted even from microdissected samples with a minimum RNA Integrity Number of 1.5. Moreover, RNA fragments up to 396 bp were assayable by reverse transcriptase-polymerase chain reaction, although short-targeted fragments as 74 bp were more consistently amplified.
CONCLUSIONS:Although variable levels of RNA fragmentation should be expected, gene expression analysis can be performed from decalcified paraffin-embedded microdissected samples, with the best results obtained for short-targeted fragments around 70 bp.</description><subject>Animals</subject><subject>Bone Demineralization Technique</subject><subject>Chelating Agents - chemistry</subject><subject>Data processing</subject><subject>Decalcification</subject><subject>Edetic Acid - chemistry</subject><subject>Embedding</subject><subject>Fixatives</subject><subject>Formaldehyde</subject><subject>Gene expression</subject><subject>Gene Expression Profiling</subject><subject>Glyceraldehyde-3-phosphate dehydrogenase</subject><subject>Glyceraldehyde-3-Phosphate Dehydrogenases - genetics</subject><subject>Glyceraldehyde-3-Phosphate Dehydrogenases - metabolism</subject><subject>Laser Capture Microdissection</subject><subject>Lasers</subject><subject>Liver</subject><subject>Liver - chemistry</subject><subject>Liver - metabolism</subject><subject>Mandible - chemistry</subject><subject>Mandible - metabolism</subject><subject>Mandibles</subject><subject>Molar - chemistry</subject><subject>Molar - metabolism</subject><subject>Molars</subject><subject>Paraffin</subject><subject>periodontal ligament</subject><subject>Periodontal Ligament - chemistry</subject><subject>Periodontal Ligament - metabolism</subject><subject>Polymerase chain reaction</subject><subject>Primers</subject><subject>Rats</subject><subject>Rats, Wistar</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>RNA</subject><subject>RNA Stability</subject><subject>RNA, Messenger - chemistry</subject><subject>RNA, Messenger - genetics</subject><subject>RNA, Messenger - isolation & purification</subject><subject>Teeth</subject><subject>Tissue Fixation</subject><issn>1052-9551</issn><issn>1533-4066</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkE1PAjEQhhujEUT_gTF79LLYz93tkQCiEaKJeN50u7Oh2v2whSD_3hrQgwfjaSaZ553JPAhdEjwkWKY3T5PFEBeYMGAkowwkk-II9YlgLOY4SY5DjwWNpRCkh868f8UYM56JU9SjVAiBpeijhxk0EE0_Ogfem7aJRo2yO298ZJpoYbRrS-M96DWU0bOqOws-qlxbRxPQympTmTBYBmQD_hydVMp6uDjUAXq5nS7Hd_H8cXY_Hs1jzTIpYs7KEotEK5oUnBc4oRKIYpqnQhcJLknJgQvFU0rTAtKq4lqTqsCpLBWvuGQDdL3f27n2Pdxd57XxGqxVDbQbnxMqSUIEp_9Agz9JpEyTgPI9Gn723kGVd87Uyu0ClH8Zz4Px_LfxELs6XNgUNZQ_oW_FAcj2wLa1a3D-zW624PIVKLte_b37E_ZCjlY</recordid><startdate>201206</startdate><enddate>201206</enddate><creator>Salmon, Cristiane Ribeiro</creator><creator>Silvério, Karina Gonzales</creator><creator>Giorgetti, Ana Paula de Oliveira</creator><creator>Sallum, Enilson Antonio</creator><creator>Casati, Márcio Zaffalon</creator><creator>Nociti, Francisco Humberto</creator><general>Lippincott Williams & Wilkins, Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>201206</creationdate><title>Gene Expression Analysis in Microdissected Samples from Decalcified Tissues</title><author>Salmon, Cristiane Ribeiro ; Silvério, Karina Gonzales ; Giorgetti, Ana Paula de Oliveira ; Sallum, Enilson Antonio ; Casati, Márcio Zaffalon ; Nociti, Francisco Humberto</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3895-43dd056ca26b44b0629e1a3c475cb60d1d4e45a47227be7ff4cc1fb079da4f493</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Animals</topic><topic>Bone Demineralization Technique</topic><topic>Chelating Agents - chemistry</topic><topic>Data processing</topic><topic>Decalcification</topic><topic>Edetic Acid - chemistry</topic><topic>Embedding</topic><topic>Fixatives</topic><topic>Formaldehyde</topic><topic>Gene expression</topic><topic>Gene Expression Profiling</topic><topic>Glyceraldehyde-3-phosphate dehydrogenase</topic><topic>Glyceraldehyde-3-Phosphate Dehydrogenases - genetics</topic><topic>Glyceraldehyde-3-Phosphate Dehydrogenases - metabolism</topic><topic>Laser Capture Microdissection</topic><topic>Lasers</topic><topic>Liver</topic><topic>Liver - chemistry</topic><topic>Liver - metabolism</topic><topic>Mandible - chemistry</topic><topic>Mandible - metabolism</topic><topic>Mandibles</topic><topic>Molar - chemistry</topic><topic>Molar - metabolism</topic><topic>Molars</topic><topic>Paraffin</topic><topic>periodontal ligament</topic><topic>Periodontal Ligament - chemistry</topic><topic>Periodontal Ligament - metabolism</topic><topic>Polymerase chain reaction</topic><topic>Primers</topic><topic>Rats</topic><topic>Rats, Wistar</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>RNA</topic><topic>RNA Stability</topic><topic>RNA, Messenger - chemistry</topic><topic>RNA, Messenger - genetics</topic><topic>RNA, Messenger - isolation & purification</topic><topic>Teeth</topic><topic>Tissue Fixation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Salmon, Cristiane Ribeiro</creatorcontrib><creatorcontrib>Silvério, Karina Gonzales</creatorcontrib><creatorcontrib>Giorgetti, Ana Paula de Oliveira</creatorcontrib><creatorcontrib>Sallum, Enilson Antonio</creatorcontrib><creatorcontrib>Casati, Márcio Zaffalon</creatorcontrib><creatorcontrib>Nociti, Francisco Humberto</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>Diagnostic molecular pathology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Salmon, Cristiane Ribeiro</au><au>Silvério, Karina Gonzales</au><au>Giorgetti, Ana Paula de Oliveira</au><au>Sallum, Enilson Antonio</au><au>Casati, Márcio Zaffalon</au><au>Nociti, Francisco Humberto</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Gene Expression Analysis in Microdissected Samples from Decalcified Tissues</atitle><jtitle>Diagnostic molecular pathology</jtitle><addtitle>Diagn Mol Pathol</addtitle><date>2012-06</date><risdate>2012</risdate><volume>21</volume><issue>2</issue><spage>120</spage><epage>126</epage><pages>120-126</pages><issn>1052-9551</issn><eissn>1533-4066</eissn><abstract>OBJECTIVE:The aim of this study was to determine the impact of standard methods for processing decalcified highly mineralized tissues on RNA yield and quality from microdissected samples.
DESIGN:Rat mandibles were fixed with either formalin-based or ethanol-based fixatives, decalcified in 20% ethylenediaminetetraacetic acid solution for 15 days, and embedded in paraffin. Transversal sections of the molars were mounted on membrane glass slides for laser capture microdissection. Unfixed frozen liver samples were used as controls to determine the impact of fixatives, decalcification and paraffin embedding on RNA integrity and recovery after sample preparation, and laser microdissection. Total RNA was obtained from periodontal ligament and fresh-frozen liver; RNA quality was assessed by Bioanalyzer, and 5 ng of total RNA was used for cDNA synthesis followed by gene expression analyses by polymerase chain reaction using 3 sets of primers for glyceraldehyde 3-phosphate dehydrogenase.
RESULTS:Data analysis demonstrated that all fixed samples presented some level of RNA fragmentation as compared with fresh-frozen samples (P<0.05). Samples fixed with Protocol (10% formalin) showed the least RNA fragmentation as compared with other fixatives (P<0.05), and biologically useful RNA was extracted even from microdissected samples with a minimum RNA Integrity Number of 1.5. Moreover, RNA fragments up to 396 bp were assayable by reverse transcriptase-polymerase chain reaction, although short-targeted fragments as 74 bp were more consistently amplified.
CONCLUSIONS:Although variable levels of RNA fragmentation should be expected, gene expression analysis can be performed from decalcified paraffin-embedded microdissected samples, with the best results obtained for short-targeted fragments around 70 bp.</abstract><cop>United States</cop><pub>Lippincott Williams & Wilkins, Inc</pub><pmid>22555095</pmid><doi>10.1097/PDM.0b013e31823e9395</doi><tpages>7</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1052-9551 |
| ispartof | Diagnostic molecular pathology, 2012-06, Vol.21 (2), p.120-126 |
| issn | 1052-9551 1533-4066 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_1291615429 |
| source | Journals@Ovid Ovid Autoload; MEDLINE |
| subjects | Animals Bone Demineralization Technique Chelating Agents - chemistry Data processing Decalcification Edetic Acid - chemistry Embedding Fixatives Formaldehyde Gene expression Gene Expression Profiling Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-Phosphate Dehydrogenases - genetics Glyceraldehyde-3-Phosphate Dehydrogenases - metabolism Laser Capture Microdissection Lasers Liver Liver - chemistry Liver - metabolism Mandible - chemistry Mandible - metabolism Mandibles Molar - chemistry Molar - metabolism Molars Paraffin periodontal ligament Periodontal Ligament - chemistry Periodontal Ligament - metabolism Polymerase chain reaction Primers Rats Rats, Wistar Reverse Transcriptase Polymerase Chain Reaction RNA RNA Stability RNA, Messenger - chemistry RNA, Messenger - genetics RNA, Messenger - isolation & purification Teeth Tissue Fixation |
| title | Gene Expression Analysis in Microdissected Samples from Decalcified Tissues |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-30T18%3A00%3A14IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-proquest_cross&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Gene%20Expression%20Analysis%20in%20Microdissected%20Samples%20from%20Decalcified%20Tissues&rft.jtitle=Diagnostic%20molecular%20pathology&rft.au=Salmon,%20Cristiane%20Ribeiro&rft.date=2012-06&rft.volume=21&rft.issue=2&rft.spage=120&rft.epage=126&rft.pages=120-126&rft.issn=1052-9551&rft.eissn=1533-4066&rft_id=info:doi/10.1097/PDM.0b013e31823e9395&rft_dat=%3Cproquest_cross%3E1013919976%3C/proquest_cross%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1013919976&rft_id=info:pmid/22555095&rfr_iscdi=true |