Quantitative analysis of interleukine-2 receptor ? mRNA expression in ankylosing sondylitis by fluorescence quantitative reverse transcription polymerase chain reaction and clinical significance
- VernacularTitle:TaqMan实时荧光定量逆转录聚合酶链反应在强直性脊柱炎白细胞介素2受体mRNA检测中的应用价值
- Author:
Fangjun LUO
;
Jian-Xin LV
;
- Publication Type:Journal Article
- Keywords:
Reverse transcription polymerase chain reaction;
Spondylitis,Ankylosing;
Receptor,interleukine-2
- From:
Chinese Journal of Laboratory Medicine
2001;0(04):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct and evaluate the real-time fluorescence quantitative polymerase chain reaction for detecting IL-2R? mRNA in ankylosing spondylitis(AS) based on TaqMan technique.Methods A pair of primers and a TaqMan probe were designed by sequence in GenBank.Total RNA isolated from the fresh peripheral blood monocytes (PBMC) of homo sapiens was amplified by the real-time FQ-RT-PCR.The product was collected by agarose gel electrophoresis and sequencing analysis then ligated with pUCm-T.The recombined plasmid was transcribed to cRNA by T7 RNA polymerase in order to prepare serial standard materials.A new method was created to quantify IL-2R? mRNA in ideal condition.Sensitivity, reproducibility and efficiency were evaluated and used, combined with sIL-2R,for clinical application of AS.Results The linear range was (7~107) cells/ml.The intra-and-inter-assay coefficient variation was 8.4% and 9.6% respectively. Recombined plasmid contained the target fragment was specific and accurate by BLAST.Standard reference was constructed successfully.The RT-PCR product in AS with HLA-B27 positive groups was higher than that with HLA-B27 negative groups and health controls (P0.05).The sensitivity of IL-2R? mRNA was 96.7%.Conclusions Real Time FQ-RT-PCR of IL-2R? mRNA is constructed successfully.This is an easy,rapid,sensitive, accurate and reliable method for quantifing IL-2R? mRNA. There is highly statistical significance, compared with sIL-2R, on the expression of IL-2R? mRNA and inflammatory states between AS and control group and effective information for administration of patients.
