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Clinical Chemistry 44: 2256-2263, 1998;
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(Clinical Chemistry. 1998;44:2256-2263.)
© 1998 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Direct reverse transcription-PCR on oligo(dT)-immobilized polypropylene microplates after capturing total mRNA from crude cell lysates

Yohei Hamaguchi1,2, Yoshimasa Aso1, Hiroshi Shimada2, and Masato Mitsuhashi1,3,a

1 Department of Pathology, University of California, Irvine, CA 92612.

2 Second Department of Surgery, Yokohama City University, School of Medicine, Yokohama 236, Japan.

3 Hitachi Chemical Research Center, 1003 Health Sciences Road West, Irvine, CA 92612.
a Author for correspondence. Fax 949-725-2727; e-mail mmitsuha{at}uci.edu.

To simplify gene expression analysis, oligo(dT)-immobilized polypropylene microplates were used serially to capture mRNA, synthesize cDNA, and amplify specific genes. The amounts of immobilized oligonucleotide, hybridized mRNA, and synthesized cDNA were quantified fluorometrically using either Yoyo-1 or AttoPhos. The immobilized oligonucleotides captured ~40–55% of mRNA directly from crude cell lysates. Hybridized mRNA was then amplified by one-step reverse transcription (RT)-PCR with rTth polymerase or two-step PCR with initial cDNA synthesis followed by PCR, where the latter exhibited more sensitivity. In two-step RT-PCR, microplates can be reused for multiple PCRs with the same or different primer sets because synthesized cDNA was covalently attached to the plates at its 5' end. We believe this microplate may be acceptable as a platform for various mRNA expression analyses, including basic research, drug screening, and molecular toxicology, as well as for molecular pathological diagnostics.




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