Effect of multiple cycles of freeze–thawing on the RNA quality of lung cancer tissues
RNA degradation is a major problem in tissue banking. We explored the effect of thawing flash-frozen biospecimens on the quality and integrity of RNA for genetic testing as well as for other cancer research studies. The histological quality of the frozen tumor sections was evaluated by using hematox...
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| Veröffentlicht in: | Cell and tissue banking 2017-09, Vol.18 (3), p.433-440 |
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| creator | Yu, Keke Xing, Jie Zhang, Jie Zhao, Ruiying Zhang, Ye Zhao, Lanxiang |
| description | RNA degradation is a major problem in tissue banking. We explored the effect of thawing flash-frozen biospecimens on the quality and integrity of RNA for genetic testing as well as for other cancer research studies. The histological quality of the frozen tumor sections was evaluated by using hematoxylin and eosin staining. RNA extraction from 60 lung cancer tissue samples subjected to various freeze/thaw cycles was performed using the RNeasy Plus isolation kit. RNA integrity was assessed by using an Agilent bioanalyzer to obtain RNA integrity numbers (RIN). Furthermore, RNA from different groups was used for fluorescence Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene mutation to verify whether it can be used for research or clinical testing. Highly variable RIN values were observed among the samples, which showed no correlation with the number of freeze/thaw cycles conducted. However, after 3 freeze/thaw cycles (each thaw event lasted for 10 min), an increasing number of changes in peak intensity in RINs were observed. After 5 freeze/thaw cycles, RNA integrity decreased to approximately 35%. After 3 freeze/thaw cycles, the RNA could still be used for RT-PCR analysis of EML4-ALK fusion gene mutations; whereas those subjected to 5 freeze/thaw cycles could not. Limited ( |
| doi_str_mv | 10.1007/s10561-016-9600-7 |
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We explored the effect of thawing flash-frozen biospecimens on the quality and integrity of RNA for genetic testing as well as for other cancer research studies. The histological quality of the frozen tumor sections was evaluated by using hematoxylin and eosin staining. RNA extraction from 60 lung cancer tissue samples subjected to various freeze/thaw cycles was performed using the RNeasy Plus isolation kit. RNA integrity was assessed by using an Agilent bioanalyzer to obtain RNA integrity numbers (RIN). Furthermore, RNA from different groups was used for fluorescence Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene mutation to verify whether it can be used for research or clinical testing. Highly variable RIN values were observed among the samples, which showed no correlation with the number of freeze/thaw cycles conducted. However, after 3 freeze/thaw cycles (each thaw event lasted for 10 min), an increasing number of changes in peak intensity in RINs were observed. After 5 freeze/thaw cycles, RNA integrity decreased to approximately 35%. After 3 freeze/thaw cycles, the RNA could still be used for RT-PCR analysis of EML4-ALK fusion gene mutations; whereas those subjected to 5 freeze/thaw cycles could not. Limited (<3) freeze/thaw cycles did not adversely affect the quality of RNA extracted from tumor tissues and subsequent RT-PCR analysis. Our data could be utilized in the establishment of a standardized procedure for tissue biospecimen collection and storage.</description><identifier>ISSN: 1389-9333</identifier><identifier>EISSN: 1573-6814</identifier><identifier>DOI: 10.1007/s10561-016-9600-7</identifier><identifier>PMID: 28573389</identifier><language>eng</language><publisher>Dordrecht: Springer Netherlands</publisher><subject>Biomedical and Life Sciences ; Biomedicine ; Cell Biology ; Data processing ; Degradation ; Fluorescence ; Freeze-thawing ; Freezing ; Fusion protein ; Genetic screening ; Life Sciences ; Lung cancer ; Lymphoma ; Point mutation ; Polymerase chain reaction ; Protein-tyrosine kinase ; Reverse transcription ; Ribonucleic acid ; RNA ; Thawing ; Tissues ; Transplant Surgery</subject><ispartof>Cell and tissue banking, 2017-09, Vol.18 (3), p.433-440</ispartof><rights>Springer Science+Business Media Dordrecht 2017</rights><rights>Cell and Tissue Banking is a copyright of Springer, 2017.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c372t-732e14db52835abe6cba51912837a09b93417d832b06cae8ad8702a8adbb8dda3</citedby><cites>FETCH-LOGICAL-c372t-732e14db52835abe6cba51912837a09b93417d832b06cae8ad8702a8adbb8dda3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s10561-016-9600-7$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s10561-016-9600-7$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27923,27924,41487,42556,51318</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28573389$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Yu, Keke</creatorcontrib><creatorcontrib>Xing, Jie</creatorcontrib><creatorcontrib>Zhang, Jie</creatorcontrib><creatorcontrib>Zhao, Ruiying</creatorcontrib><creatorcontrib>Zhang, Ye</creatorcontrib><creatorcontrib>Zhao, Lanxiang</creatorcontrib><title>Effect of multiple cycles of freeze–thawing on the RNA quality of lung cancer tissues</title><title>Cell and tissue banking</title><addtitle>Cell Tissue Bank</addtitle><addtitle>Cell Tissue Bank</addtitle><description>RNA degradation is a major problem in tissue banking. We explored the effect of thawing flash-frozen biospecimens on the quality and integrity of RNA for genetic testing as well as for other cancer research studies. The histological quality of the frozen tumor sections was evaluated by using hematoxylin and eosin staining. RNA extraction from 60 lung cancer tissue samples subjected to various freeze/thaw cycles was performed using the RNeasy Plus isolation kit. RNA integrity was assessed by using an Agilent bioanalyzer to obtain RNA integrity numbers (RIN). Furthermore, RNA from different groups was used for fluorescence Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene mutation to verify whether it can be used for research or clinical testing. Highly variable RIN values were observed among the samples, which showed no correlation with the number of freeze/thaw cycles conducted. However, after 3 freeze/thaw cycles (each thaw event lasted for 10 min), an increasing number of changes in peak intensity in RINs were observed. After 5 freeze/thaw cycles, RNA integrity decreased to approximately 35%. After 3 freeze/thaw cycles, the RNA could still be used for RT-PCR analysis of EML4-ALK fusion gene mutations; whereas those subjected to 5 freeze/thaw cycles could not. Limited (<3) freeze/thaw cycles did not adversely affect the quality of RNA extracted from tumor tissues and subsequent RT-PCR analysis. Our data could be utilized in the establishment of a standardized procedure for tissue biospecimen collection and storage.</description><subject>Biomedical and Life Sciences</subject><subject>Biomedicine</subject><subject>Cell Biology</subject><subject>Data processing</subject><subject>Degradation</subject><subject>Fluorescence</subject><subject>Freeze-thawing</subject><subject>Freezing</subject><subject>Fusion protein</subject><subject>Genetic screening</subject><subject>Life Sciences</subject><subject>Lung cancer</subject><subject>Lymphoma</subject><subject>Point mutation</subject><subject>Polymerase chain reaction</subject><subject>Protein-tyrosine kinase</subject><subject>Reverse transcription</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>Thawing</subject><subject>Tissues</subject><subject>Transplant Surgery</subject><issn>1389-9333</issn><issn>1573-6814</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp1kM9KxDAQxoMo_n8AL1Lw4qU6SbZNchTxH4iCKB5Dmk7XSrddkxZZT76Db-iTOMuuIoKnSeb7zXzJx9gehyMOoI4jhyznKfA8NTlAqlbYJs-UTHPNR6t0ltqkRkq5wbZifAYQoIRcZxtCE0XiJns8qyr0fdJVyWRo-nraYOJnvsE4b1UB8Q0_3z_6J_dat-Oka5P-CZO7m5PkZXBN3c_mWDOQ5F3rMSR9HeOAcYetVa6JuLus2-zh_Oz-9DK9vr24Oj25Tr1Uok-VFMhHZZEJLTNXYO4Ll3HD6aocmMLIEVellqKA3DvUrtQKhKNaFLosndxmh4u909C9kG9vJ3X02DSuxW6IlhugrxrQQOjBH_S5G0JLryNK5uSpTU4UX1A-dDEGrOw01BMXZpaDnaduF6lbSt3OU7eKZvaXm4diguXPxHfMBIgFEElqxxh-Wf-79QsRoI0L</recordid><startdate>20170901</startdate><enddate>20170901</enddate><creator>Yu, Keke</creator><creator>Xing, Jie</creator><creator>Zhang, Jie</creator><creator>Zhao, Ruiying</creator><creator>Zhang, Ye</creator><creator>Zhao, Lanxiang</creator><general>Springer Netherlands</general><general>Springer Nature B.V</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7RV</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>8AO</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB0</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>NAPCQ</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope></search><sort><creationdate>20170901</creationdate><title>Effect of multiple cycles of freeze–thawing on the RNA quality of lung cancer tissues</title><author>Yu, Keke ; Xing, Jie ; Zhang, Jie ; Zhao, Ruiying ; Zhang, Ye ; Zhao, Lanxiang</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c372t-732e14db52835abe6cba51912837a09b93417d832b06cae8ad8702a8adbb8dda3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Biomedical and Life Sciences</topic><topic>Biomedicine</topic><topic>Cell Biology</topic><topic>Data processing</topic><topic>Degradation</topic><topic>Fluorescence</topic><topic>Freeze-thawing</topic><topic>Freezing</topic><topic>Fusion protein</topic><topic>Genetic screening</topic><topic>Life Sciences</topic><topic>Lung cancer</topic><topic>Lymphoma</topic><topic>Point mutation</topic><topic>Polymerase chain reaction</topic><topic>Protein-tyrosine kinase</topic><topic>Reverse transcription</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>Thawing</topic><topic>Tissues</topic><topic>Transplant Surgery</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Yu, Keke</creatorcontrib><creatorcontrib>Xing, Jie</creatorcontrib><creatorcontrib>Zhang, Jie</creatorcontrib><creatorcontrib>Zhao, Ruiying</creatorcontrib><creatorcontrib>Zhang, Ye</creatorcontrib><creatorcontrib>Zhao, Lanxiang</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Nursing & Allied Health Database</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Nursing & Allied Health Premium</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><jtitle>Cell and tissue banking</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Yu, Keke</au><au>Xing, Jie</au><au>Zhang, Jie</au><au>Zhao, Ruiying</au><au>Zhang, Ye</au><au>Zhao, Lanxiang</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Effect of multiple cycles of freeze–thawing on the RNA quality of lung cancer tissues</atitle><jtitle>Cell and tissue banking</jtitle><stitle>Cell Tissue Bank</stitle><addtitle>Cell Tissue Bank</addtitle><date>2017-09-01</date><risdate>2017</risdate><volume>18</volume><issue>3</issue><spage>433</spage><epage>440</epage><pages>433-440</pages><issn>1389-9333</issn><eissn>1573-6814</eissn><abstract>RNA degradation is a major problem in tissue banking. We explored the effect of thawing flash-frozen biospecimens on the quality and integrity of RNA for genetic testing as well as for other cancer research studies. The histological quality of the frozen tumor sections was evaluated by using hematoxylin and eosin staining. RNA extraction from 60 lung cancer tissue samples subjected to various freeze/thaw cycles was performed using the RNeasy Plus isolation kit. RNA integrity was assessed by using an Agilent bioanalyzer to obtain RNA integrity numbers (RIN). Furthermore, RNA from different groups was used for fluorescence Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene mutation to verify whether it can be used for research or clinical testing. Highly variable RIN values were observed among the samples, which showed no correlation with the number of freeze/thaw cycles conducted. However, after 3 freeze/thaw cycles (each thaw event lasted for 10 min), an increasing number of changes in peak intensity in RINs were observed. After 5 freeze/thaw cycles, RNA integrity decreased to approximately 35%. After 3 freeze/thaw cycles, the RNA could still be used for RT-PCR analysis of EML4-ALK fusion gene mutations; whereas those subjected to 5 freeze/thaw cycles could not. Limited (<3) freeze/thaw cycles did not adversely affect the quality of RNA extracted from tumor tissues and subsequent RT-PCR analysis. Our data could be utilized in the establishment of a standardized procedure for tissue biospecimen collection and storage.</abstract><cop>Dordrecht</cop><pub>Springer Netherlands</pub><pmid>28573389</pmid><doi>10.1007/s10561-016-9600-7</doi><tpages>8</tpages></addata></record> |
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| ispartof | Cell and tissue banking, 2017-09, Vol.18 (3), p.433-440 |
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| subjects | Biomedical and Life Sciences Biomedicine Cell Biology Data processing Degradation Fluorescence Freeze-thawing Freezing Fusion protein Genetic screening Life Sciences Lung cancer Lymphoma Point mutation Polymerase chain reaction Protein-tyrosine kinase Reverse transcription Ribonucleic acid RNA Thawing Tissues Transplant Surgery |
| title | Effect of multiple cycles of freeze–thawing on the RNA quality of lung cancer tissues |
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