A faithful method for PCR-mediated global mRNA amplification and its integration into microarray analysis on laser-captured cells.
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.
Aoyagi
Kazuhiko
K
Genetics Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chrome, Chuo-ku, Tokyo 104-0045, Japan. kaaoyagi@gan2.res.ncc.go.jp
Tatsuta
Takeshi
T
Nishigaki
Michiko
M
Akimoto
Shingo
S
Tanabe
Chikako
C
Omoto
Yoko
Y
Hayashi
Shin ichi
Si
Sakamoto
Hiromi
H
Sakamoto
Michiie
M
Yoshida
Teruhiko
T
Terada
Masaaki
M
Sasaki
Hiroki
H
D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D006801:Humans; D007834:Lasers; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D018075:RNA, Complementary / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D013274:Stomach Neoplasms / Q000235:genetics / Q000473:pathology; D014407:Tumor Cells, Cultured
D018075:RNA, Complementary; D012333:RNA, Messenger