Differential Gene Expression Analysis Using Paraffin-Embedded Tissues after Laser Microdissection

Joung Ok Kim, Hyun Nam Kim, Mi Hye Hwang, Hong In Shin, Shin Yoon Kim, Rang Woon Park, Eui Yun Park, In San Kim, Andre J. Van Wijnen, Janet L. Stein, Jane B. Lian, Gary S. Stein, Je Yong Choi

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31 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)998-1006
Number of pages9
JournalJournal of cellular biochemistry
Volume90
Issue number5
DOIs
Publication statusPublished - 2003 Dec 1

Keywords

  • Chondrocyte
  • Laser microdissection
  • Methacarn
  • Paraffin
  • RNA

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Kim, J. O., Kim, H. N., Hwang, M. H., Shin, H. I., Kim, S. Y., Park, R. W., Park, E. Y., Kim, I. S., Van Wijnen, A. J., Stein, J. L., Lian, J. B., Stein, G. S., & Choi, J. Y. (2003). Differential Gene Expression Analysis Using Paraffin-Embedded Tissues after Laser Microdissection. Journal of cellular biochemistry, 90(5), 998-1006. https://doi.org/10.1002/jcb.10680