抄録
In this report, we have developed a novel quantitative RT-PCR protocol in which the procedure including mRNA purification can be performed in an all-in-one tube. To simplify gene expression analysis, oligo-dT30 immobilized PCR tubes were used serially to capture mRNA, synthesize solid-phase cDNA, and amplify specific genes. The immobilized oligo-dT30 can efficiently capture mRNA directly from crude human cell lysates. The captured mRNA is then amplified by one-step reverse transcription PCR (RT-PCR) with initial cDNA synthesis followed by PCR. In RT-PCR, this new reusable PCR tube device can be employed for multiple PCR amplifications with different primer sets from a solid-phase oligo-dT30 primed cDNA library. This paper introduces a novel and highly reliable all-in-one tube method for rapid cell lysis, followed by quantitative preparation and expression analysis of target mRNA molecules with small amounts of sample. This procedure allows all steps to be carried out by sequential dilution in a single tube, without chemical extraction. We demonstrate the utility of this novel method by quantification of two housekeeping genes, β-actin and GAPDH, in HeLa cells. We believe this new PCR device can be useful as a platform for various mRNA expression analyses, including basic research, drug screening, and molecular toxicology, as well as for molecular pathological diagnostics.
