Clinical Chemistry
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Clinical Chemistry 43: 764-770, 1997;
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(Clinical Chemistry. 1997;43:764-770.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Construction of cDNA bank from biopsy specimens for multiple gene analysis of cancer

Takashi Ishikawa1, Yasushi Ichikawa1, Yasuhiko Miura1, Mobuyoshi Momiyama1, Cylia Keller2, Kenneth Koo2, Tatsuo Akitaya3, Hiroshi Shimada1 and Masato Mitsuhashi2,a

1 The Second Department of Surgery, Yokohama City University, School of Medicine, Yokohama, Japan.

2 Hitachi Chemical Research Center, 1003 Health Sciences Rd. West, Irvine, CA 92612.

3 Hitachi Chemical Co., Ltd., Ibaraki, Japan.
a Author for correspondence. Fax 714-725-2727; e-mail HCN02644{at}niftyserve.or.jp

We constructed a "cDNA bank" of human colorectal cancer and surrounding normal tissues with our unique mRNA assay system. Total nucleic acids extracted from patients' tissues were applied to 96-well plates, where poly(dT) sequences of oligonucleotides were immobilized. After hybridization, the cDNA was reverse-transcribed on the plate with the captured mRNA as a template, followed by synthesis of double-stranded (ds) cDNA. The resulting sense cDNA was removed from the plate, then used in PCR for analysis of various genes. The sense strand of the cDNA was repeatedly synthesized by using the immobilized antisense cDNA as a template even from plates used once and stored at 4 °C for as long as 6 months. Furthermore, the results of PCR could be easily compared among different specimens if the same amount of total mRNA were applied to the plate for the ds cDNA synthesis. This demonstrated that the cDNA bank constructed from clinical materials provides almost unlimited supplies of cDNA for multiple gene analysis of cancer.


Key Words: indexing terms: mRNA • reverse transcription–polymerase chain reaction • oligonucleotides




The following articles in journals at HighWire Press have cited this article:


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 Clin. Chem.Home page
M. Mitsuhashi, S. Tomozawa, K. Endo, and A. Shinagawa
Quantification of mRNA in Whole Blood by Assessing Recovery of RNA and Efficiency of cDNA Synthesis
Clin. Chem., April 1, 2006; 52(4): 634 - 642.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
Y. Hamaguchi, Y. Aso, H. Shimada, and M. Mitsuhashi
Direct reverse transcription-PCR on oligo(dT)-immobilized polypropylene microplates after capturing total mRNA from crude cell lysates
Clin. Chem., November 1, 1998; 44(11): 2256 - 2263.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
K. Eguchi, Y. Hamaguchi, Y. Aso, T. Shioiri, M. Ogura, and M. Mitsuhashi
Oligo(dT)-immobilized Pipette Tip: Efficient New Methodology for mRNA Preparation and Direct Gene Amplification
Clin. Chem., October 1, 1998; 44(10): 2208 - 2210.
[Full Text] [PDF]


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