The measurement of transforming growth factor ?1 mRNA in peripheral blood mononuclear cell of patients with chronic hepatic disease by fluorescence quantitative reverse transcription polymerase chain reaction
- VernacularTitle:荧光定量逆转录聚合酶链反应检测慢性肝病患者单个核细胞中TGF-?1的表达水平
- Author:
Rong-Mei DING
;
Jian-Xin LV
;
- Publication Type:Journal Article
- Keywords:
Reverse transcription polymerase chain reaction;
Transforming growth factor ?1;
Hepatic fibrosis
- From:
Chinese Journal of Laboratory Medicine
2003;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop fluorescence quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) for detection and quantitation of transforming growth factor ?1 (TGF-?1) mRNA in peripheral blood mononuclear cell (PBMC) of patients with chronic hepatic disease.Methods The TGF-?1 recombined plasmid was constructed by conventional molecular biological techniques,and strand-specific cRNA standard was synthesized by T7 RNA polymerase in vitro transcription.The cRNA standard and a TaqMan-MGB probe were used for quantitation of the TGF-?1 mRNA,and the assay was evaluated.Moreover,the plasma TGF-?1 was detected by ELISA.Results Sequence analysis indicated that the recombined plasmid contains the specific 102bp fragment of TGF-?1. The FQ-RT-PCR could detect as low as 6.81 copies of standard cRNA,the linear range was from 6.81 to 6.81?10~9 copies,and the coefficient variation was 1.28%-2.27% and 2.56%-2.61% respectively in intra and inter-assay.The levels of TGF-?1 mRNA in PBMC and plasma TGF-?1 of patients with chronic hepatic disease were significantly higher than that of healthy controls(P
