39. Expression Profile Using HiCEP

Masumi Abe, Ryutaro Fukumura, Tatsuya Ohhata, Ryoko Araki, Yuki Miyamoto, Can Ismail, Hajime Ohkaze, Hirokazu Takahashi, Yoko Tsutsumi, Yuko Hoki, Akira Fujimori and Toshiyuki Saito

Keywords: transcripts, expression profile, mouse embryonic fibroblast, X-ray irradiation

Cells save themselves and/or their hosts under stressful conditions through various mechanisms, one of which is programmed expression of a subset of genes. A novel technique exhibiting all transcriptional events in a cell would provide a great tool to understand such important mechanisms in life as differentiation and apoptosis. We have previously developed an expression profile system that can detect approximately 80% of the entire transcripts from a minimal size of cell or tissue samples (HiCEP).

This year, we investigated the sensitivity and the reproducibility of this novel strategy. (1) Different amounts of synthesized poly(A)RNA were added to total RNA from budding yeast and a test for detection by HiCEP was performed. The result showed that HiCEP is so sensitive as to be able to detect one copy per cell of the contaminated poly(A)RNA. (2) We performed HiCEP analyses for mouse embryonic fibroblast (MEF) cell samples, which were harvested at 0, 3, 6 and 24 hr after X-ray irradiation (7Gy). More than 200 transcripts exhibited altered levels of expression among approx. 20,000 transcripts detected in the HiCEP analysis. Those included p21 gene, for which expression increased 2-fold in the first 3 hrs after irradiation. Quantitative RT-PCR targeting each individual transcript verified that alteration of expression level by 1.3-fold was reliable in the HiCEP analyses.

We also performed expression analysis with MEF's of mice that were deficient in p53 or in ATM. Among 200 of the X-ray associated transcripts, only a subset of the genes was altered in both types of knockout mice, while the majority of them appeared to depend on either p53 or ATM.

Recent development of extensive genome resources is facilitating establishment of a database for the HiCEP analysis. We are now developing software to handle a great amount of HiCEP data more efficiently in order to realize bioinformatics on the HiCEP database. In the future, our HiCEP database should greatly contribute to medical, pharmaceutical and agricultural applications.

Publications:
Fukumura R., Takahashi H., Saito T., Sujino M., Umeda N., Inouye S.T., Tsutsumi Y., Fujimori A., Sato S., Tatsumi K., Araki R. and Abe M. A wide-coverage, highly sensitive gene expression profiling procedure applicable to all eukaryotes. Submitted for publication.