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.
|Biochemical and Biophysical Research Communications
|Published - 2003 1月 24
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