Show simple item record

dc.contributor.authorKim, Joung-Ok
dc.contributor.authorKim, Hyun-Nam
dc.contributor.authorHwang, Mi-Hye
dc.contributor.authorShin, Hong-In
dc.contributor.authorKim, Shin-Yoon
dc.contributor.authorPark, Rang-Woon
dc.contributor.authorPark, Eui-Yun
dc.contributor.authorKim, In-San
dc.contributor.authorVan Wijnen, Andre J.
dc.contributor.authorStein, Janet L.
dc.contributor.authorLian, Jane B.
dc.contributor.authorStein, Gary S.
dc.contributor.authorChoi, Je-Yong
dc.date2022-08-11T08:08:59.000
dc.date.accessioned2022-08-23T16:14:49Z
dc.date.available2022-08-23T16:14:49Z
dc.date.issued2003-11-19
dc.date.submitted2008-10-15
dc.identifier.citationJ Cell Biochem. 2003 Dec 1;90(5):998-1006. <a href="http://dx.doi.org/10.1002/jcb.10680 ">Link to article on publisher's site</a>
dc.identifier.issn0730-2312 (Print)
dc.identifier.doi10.1002/jcb.10680
dc.identifier.pmid14624459
dc.identifier.urihttp://hdl.handle.net/20.500.14038/33950
dc.description.abstractRecent advances in laser microdissection allow for precise removal of pure cell populations from morphologically preserved tissue sections. However, RNA from paraffin-embedded samples is usually degraded during microdissection. The purpose of this study is to determine the optimal fixative for RNA extractions from laser microdissected paraffin-embedded samples. The integrity of RNA was evaluated with the intactness of 18S and 28S ribosomal RNA by electrophoresis and by the length of individual gene transcripts using RT-PCR. The various fixatives were methacarn (a combination of methanol, chloroform, and acetic acid) and several concentrations of ethanol and isopropanol. Methacarn was the optimal fixative for RNA preservation in paraffin-embedded tissues, which included liver, lung, kidney, muscle, and limb. Based on RT-PCR analysis, methacarn fixed samples exhibited the expected RNA sizes for individual genes such as glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and bone-related genes (e.g., alkaline phosphatase and osteonectin). The laser microdissection technique with methacarn fixation was then applied to analyze the differential gene expression between hypertrophic and proliferative chondrocytes in the growth plate of long bone. The expression of type X collagen, a specific gene for hypertrophic chondrocytes, was only observed in hypertrophic chondrocytes, while type II collagen was observed more broadly in the growth plate as anticipated. Thus, combining laser microdissection with methacarn fixation facilitates the examination of differentially expressed genes from various tissues.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14624459&dopt=Abstract">Link to article in PubMed</a>
dc.relation.urlhttp://dx.doi.org/10.1002/jcb.10680
dc.subjectAcetic Acid; Animals; Chloroform; Dissection; Fixatives; *Gene Expression Profiling; Humans; *Lasers; Methanol; Mice; Mice, Inbred ICR; *Paraffin Embedding; RNA; Reverse Transcriptase Polymerase Chain Reaction; *Tissue Fixation
dc.subjectLife Sciences
dc.subjectMedicine and Health Sciences
dc.titleDifferential gene expression analysis using paraffin-embedded tissues after laser microdissection
dc.typeJournal Article
dc.source.journaltitleJournal of cellular biochemistry
dc.source.volume90
dc.source.issue5
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_sp/602
dc.identifier.contextkey651072
html.description.abstract<p>Recent advances in laser microdissection allow for precise removal of pure cell populations from morphologically preserved tissue sections. However, RNA from paraffin-embedded samples is usually degraded during microdissection. The purpose of this study is to determine the optimal fixative for RNA extractions from laser microdissected paraffin-embedded samples. The integrity of RNA was evaluated with the intactness of 18S and 28S ribosomal RNA by electrophoresis and by the length of individual gene transcripts using RT-PCR. The various fixatives were methacarn (a combination of methanol, chloroform, and acetic acid) and several concentrations of ethanol and isopropanol. Methacarn was the optimal fixative for RNA preservation in paraffin-embedded tissues, which included liver, lung, kidney, muscle, and limb. Based on RT-PCR analysis, methacarn fixed samples exhibited the expected RNA sizes for individual genes such as glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and bone-related genes (e.g., alkaline phosphatase and osteonectin). The laser microdissection technique with methacarn fixation was then applied to analyze the differential gene expression between hypertrophic and proliferative chondrocytes in the growth plate of long bone. The expression of type X collagen, a specific gene for hypertrophic chondrocytes, was only observed in hypertrophic chondrocytes, while type II collagen was observed more broadly in the growth plate as anticipated. Thus, combining laser microdissection with methacarn fixation facilitates the examination of differentially expressed genes from various tissues.</p>
dc.identifier.submissionpathgsbs_sp/602
dc.contributor.departmentDepartment of Cell Biology
dc.contributor.departmentGraduate School of Biomedical Sciences
dc.source.pages998-1006


This item appears in the following Collection(s)

Show simple item record