Differential gene expression analysis using paraffin-embedded tissues after laser microdissection
Biochemistry & Molecular Pharmacology
Graduate School of Biomedical Sciences; Department of Cell Biology
Medical Subject Headings
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 Fixation
Life Sciences | Medicine and Health Sciences
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.
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Citation: J Cell Biochem. 2003 Dec 1;90(5):998-1006. Link to article on publisher's site
Kim, Joung-Ok; Kim, 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.; and Choi, Je-Yong, "Differential gene expression analysis using paraffin-embedded tissues after laser microdissection" (2003). GSBS Student Publications. Paper 602.