| RRC ID |
42643
|
| 著者 |
Oba R, Kudo Y, Sato N, Noda R, Otsuka Y.
|
| タイトル |
A new method of competitive reverse transcription polymerase chain reaction with SYBR Gold staining for quantitative analysis of mRNA.
|
| ジャーナル |
Electrophoresis
|
| Abstract |
There are several methods available to analyze the mRNA concentration quantitatively. Among them, the competitive reverse transcription (RT-)PCR method is very useful. For this method, Cy5-labeled primers were used, and after gel electrophoresis in 7 M urea, the Cy5-labeled single-strand DNA was measured by a fluorescence detector. However, as the equipment to measure the Cy5-labeled fluorescence is expensive, we developed a new method using SYBR Gold staining. After gel electrophoresis in 7 M urea, the single-strand PCR product DNA was stained with SYBR Gold, and photographed with a standard UV-transilluminator and a standard digital camera with a specific filter for SYBR Gold staining. The photographic image was digitized by an imaging software. We measured beta-actin and plasma glutathione peroxidase (Gpx3) mRNA concentrations of HepG2 cell cultured at 5 and 20% oxygen tension. The Gpx3 expression was increased by hypoxia. The result was equivalent to the data obtained by the real-time PCR analysis.
|
| 巻・号 |
27(14)
|
| ページ |
2865-8
|
| 公開日 |
2006-7-1
|
| DOI |
10.1002/elps.200500568
|
| PMID |
16688698
|
| MeSH |
Actins / genetics
Carbocyanines / chemistry
Cells, Cultured
DNA Primers / chemistry
DNA, Single-Stranded / analysis
DNA, Single-Stranded / chemistry
Fluorescent Dyes / chemistry
Glutathione Peroxidase / genetics
Humans
Organic Chemicals / chemistry*
RNA, Messenger / analysis*
Reverse Transcriptase Polymerase Chain Reaction / methods*
|
| IF |
3.081
|
| 引用数 |
3
|
|
WOS 分野
|
CHEMISTRY, ANALYTICAL
BIOCHEMICAL RESEARCH METHODS
|
| リソース情報 |
| ヒト・動物細胞 |
|
