All-in-one tube method for quantitative gene expression analysis in oligo-dT(30) immobilized PCR tube coated with MPC polymer.

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-dT(30) immobilized PCR tubes were used serially to capture mRNA, synthesize solid-phase cDNA, and amplify specific genes. The immobilized oligo-dT(30) 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-dT(30) 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, beta-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.

Multiple primer extension by DNA polymerase on a novel plastic DNA array coated with a biocompatible polymer.

DNA microarrays are routinely used to monitor gene expression profiling and single nucleotide polymorphisms (SNPs). However, for practically useful high performance, the detection sensitivity is still not adequate, leaving low expression genes undetected. To resolve this issue, we have developed a new plastic S-BIO PrimeSurface with a biocompatible polymer; its surface chemistry offers an extraordinarily stable thermal property for a lack of pre-activated glass slide surface. The oligonucleotides immobilized on this substrate are robust in boiling water and show no significant loss of hybridization activity during dissociation treatment. This allowed us to hybridize the templates, extend the 3' end of the immobilized DNA primers on the S-Bio by DNA polymerase using deoxynucleotidyl triphosphates (dNTP) as extender units, release the templates by denaturalization and use the same templates for a second round of reactions similar to that of the PCR method. By repeating this cycle, the picomolar concentration range of the template oligonucleotide can be detected as stable signals via the incorporation of labeled dUTP into primers. This method of Multiple Primer EXtension (MPEX) could be further extended as an alternative route for producing DNA microarrays for SNP analyses via simple template preparation such as reverse transcript cDNA or restriction enzyme treatment of genome DNA.