| RRC ID |
10933
|
| 著者 |
Moriya Y, Nakamura T, Okamura N, Sakaeda T, Horinouchi M, Tamura T, Aoyama N, Kasuga M, Okumura K.
|
| タイトル |
Comparison of synthetic DNA templates with authentic cDNA templates in terms of quantification by real-time quantitative reverse transcription polymerase chain reaction.
|
| ジャーナル |
Biol Pharm Bull
|
| Abstract |
Synthetic DNA templates were compared with authentic cDNA templates as standards for the real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The single-stranded DNA template used here targeted the multidrug resistant transporter P-glycoprotein/MDR1. The double-stranded DNA template, targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was synthesized using an exonuclease-free large fragment E. coli DNA polymerase I. The human colon carcinoma cell line Caco-2 and human duodenum biopsies were used to prepare the authentic cDNA templates. The standard lines were comparable for the synthetic DNA templates and authentic cDNA templates. Long-term cryopreservation at -80 degrees C resulted in the destabilization of the synthetic single-stranded DNA template compared with the authentic cDNA templates in the case of MDR1, whereas for GAPDH, the stability of the synthetic double-stranded DNA template was comparable with that of the authentic cDNA templates. Even for the synthetic DNA templates, repetitive freeze-thawing resulted in destabilization, especially at lower concentrations, and degradation products might have interfered with the RT-PCR's efficiency. The synthetic DNA templates are better than the authentic cDNA templates, but more than 5 cycles of repetitive freeze-thawing should be avoided.
|
| 巻・号 |
29(3)
|
| ページ |
535-8
|
| 公開日 |
2006-3-1
|
| DOI |
10.1248/bpb.29.535
|
| PII |
JST.JSTAGE/bpb/29.535
|
| PMID |
16508161
|
| MeSH |
ATP Binding Cassette Transporter, Subfamily B, Member 1 / biosynthesis
ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism
Caco-2 Cells
Cryopreservation
DNA / chemical synthesis*
DNA / chemistry*
DNA, Complementary / chemistry*
DNA, Neoplasm / chemistry
Data Interpretation, Statistical
Duodenum / chemistry
Freezing
Glyceraldehyde-3-Phosphate Dehydrogenases / chemistry
Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism
Humans
Male
Oligonucleotides / chemical synthesis
Oligonucleotides / chemistry
Reproducibility of Results
Reverse Transcriptase Polymerase Chain Reaction
Templates, Genetic*
|
| IF |
1.863
|
| 引用数 |
13
|
|
WOS 分野
|
PHARMACOLOGY & PHARMACY
|
| リソース情報 |
| ヒト・動物細胞 |
CACO-2(RCB0988) |
