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.
Tanaka
Atsuko
A
School of Pharmaceutical Sciences, Mukogawa Women's University, Koshien, Nishinomiya 663-8179, Japan.
Harikai
Naoki
N
Saito
Shin
S
Yakabe
Toru
T
Funaoka
Sohei
S
Yokoyama
Kanehisa
K
Fujiwara
Kazuhiko
K
Iwao-Koizumi
Kyoko
K
Murata
Shigenori
S
Kinoshita
Kenji
K
D000199:Actins / Q000235:genetics; D020869:Gene Expression Profiling / Q000295:instrumentation* / Q000379:methods; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008722:Methods; D009838:Oligodeoxyribonucleotides; D011108:Polymers; D012333:RNA, Messenger / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation
D000199:Actins; D009838:Oligodeoxyribonucleotides; D011108:Polymers; D012333:RNA, Messenger; C027903:oligo (dT); D005987:Glyceraldehyde-3-Phosphate Dehydrogenases