Differential gene expression analysis using paraffin-embedded tissues after laser microdissection
Authors
Kim, Joung-OkKim, Hyun-Nam
Hwang, Mi-Hye
Shin, Hong-In
Kim, Shin-Yoon
Park, Rang-Woon
Park, Eui-Yun
Kim, In-San
Van Wijnen, Andre J.
Stein, Janet L.
Lian, Jane B.
Stein, Gary S.
Choi, Je-Yong
Document Type
Journal ArticlePublication Date
2003-11-19Keywords
Acetic Acid; Animals; Chloroform; Dissection; Fixatives; *Gene Expression Profiling; Humans; *Lasers; Methanol; Mice; Mice, Inbred ICR; *Paraffin Embedding; RNA; Reverse Transcriptase Polymerase Chain Reaction; *Tissue FixationLife Sciences
Medicine and Health Sciences
Metadata
Show full item recordAbstract
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.Source
J Cell Biochem. 2003 Dec 1;90(5):998-1006. Link to article on publisher's siteDOI
10.1002/jcb.10680Permanent Link to this Item
http://hdl.handle.net/20.500.14038/33950PubMed ID
14624459Related Resources
Link to article in PubMedae974a485f413a2113503eed53cd6c53
10.1002/jcb.10680