A faithful method for PCR-mediated global mRNA amplification and its integration into microarray analysis on laser-captured cells

Kazuhiko Aoyagi, Takeshi Tatsuta, Michiko Nishigaki, Shingo Akimoto, Chikako Tanabe, Yoko Omoto, Shin ichi Hayashi, Hiromi Sakamoto, Michiie Sakamoto, Teruhiko Yoshida, Masaaki Terada, Hiroki Sasaki

Research output: Contribution to journalArticle

80 Citations (Scopus)

Abstract

Quantitative and qualitative analyses of mRNAs from a small number of cells are extremely important for studies on gene expression in various physiological and pathological conditions in multicellular organisms. We present here an effective method for high-fidelity global mRNA amplification for in vivo gene expression profiling of as few as 100 cells obtained by laser-captured microdissection (LCM). This method, called TALPAT, is based on T7 RNA polymerase-mediated transcription, adaptor ligation, and PCR amplification followed by T7-transcription. More than 80% of genes were commonly identified as a more than 3-fold changed gene among three gastric cancer cell lines using cRNA amplified by both TALPAT and the ordinary in vitro T7-transcription. The reproducibility of TALPAT was validated by microarray analysis on 100 breast cancer cells obtained by LCM. For the application of the LCM-TALPAT method, we successfully obtained expression profiles of gastric cancer cells and the mesenchymal cells, enabling us to understand in vivo cell-to-cell cross-talk in the microenvironment.

Original languageEnglish
Pages (from-to)915-920
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume300
Issue number4
DOIs
Publication statusPublished - 2003 Jan 24
Externally publishedYes

Fingerprint

Microdissection
Transcription
Microarray Analysis
Microarrays
Amplification
Lasers
Cells
Gene expression
Polymerase Chain Reaction
Messenger RNA
Genes
Complementary RNA
Stomach Neoplasms
Gene Expression Profiling
Ligation
Cell Count
Breast Neoplasms
Gene Expression
Cell Line

Keywords

  • Breast cancer
  • Expression profiling
  • Gastric cancer
  • Global mRNA amplification
  • LCM
  • Microarray

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

A faithful method for PCR-mediated global mRNA amplification and its integration into microarray analysis on laser-captured cells. / Aoyagi, Kazuhiko; Tatsuta, Takeshi; Nishigaki, Michiko; Akimoto, Shingo; Tanabe, Chikako; Omoto, Yoko; Hayashi, Shin ichi; Sakamoto, Hiromi; Sakamoto, Michiie; Yoshida, Teruhiko; Terada, Masaaki; Sasaki, Hiroki.

In: Biochemical and Biophysical Research Communications, Vol. 300, No. 4, 24.01.2003, p. 915-920.

Research output: Contribution to journalArticle

Aoyagi, K, Tatsuta, T, Nishigaki, M, Akimoto, S, Tanabe, C, Omoto, Y, Hayashi, SI, Sakamoto, H, Sakamoto, M, Yoshida, T, Terada, M & Sasaki, H 2003, 'A faithful method for PCR-mediated global mRNA amplification and its integration into microarray analysis on laser-captured cells', Biochemical and Biophysical Research Communications, vol. 300, no. 4, pp. 915-920. https://doi.org/10.1016/S0006-291X(02)02967-4
Aoyagi, Kazuhiko ; Tatsuta, Takeshi ; Nishigaki, Michiko ; Akimoto, Shingo ; Tanabe, Chikako ; Omoto, Yoko ; Hayashi, Shin ichi ; Sakamoto, Hiromi ; Sakamoto, Michiie ; Yoshida, Teruhiko ; Terada, Masaaki ; Sasaki, Hiroki. / A faithful method for PCR-mediated global mRNA amplification and its integration into microarray analysis on laser-captured cells. In: Biochemical and Biophysical Research Communications. 2003 ; Vol. 300, No. 4. pp. 915-920.
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