基于通用质粒标准品的实时荧光定量PCR检测组织因子剪接异构体F3tv1及F3tv2

Quantitative determination of human tissue factor spliced variants F3tv1 and F3tv2 by real-time PCR with universal plasmid standard

目的建立基于通用质粒标准品的实时荧光定量PCR(qPCR)法检测人组织因子(TF)两种剪接异构体(F3tv1和F3tv2)。方法设计基因特异性上游引物与通用下游引物,通过引物末端延伸法将2种扩增产物进行序列简并,用于构建通用质粒标准品。用qPCR法检测人白血病细胞株THP-1、Jurkat及外周血单核细胞中F3tv1与F3tv2的表达,分析该法的线性范围与特异性。结果所建qPCR方法对F3tv1与F3tv2扩增特异,线性范围均为108～101copies/μL。THP-1与外周血单核细胞中F3tv1相对表达量分别为(5.70×10-4)±(2.62×10-5)与(1.79×10-4)±(4.37×10-5);F3tv2分别为(4.94×10-5)±(2.19×10-6)与(2.98±2.18)×10-6,Jurkat细胞株F3tv1与F3tv2均未检出。结论建立的qPCR方法可对TF两种剪接异构体同时进行精确、定量地检测,为深入研究TF的选择性剪接调控机制提供实验依据。

Objective To establish a real-time PCR with universal plasmid standard for quantitative determination of human full-length and alternative spliced tissue factor transcripts F3tvl, F3tv2. Methods The gene-specific forward primers and universal reverse primer were designed. The degeneracy of the sequences aligned from amplicons of F3tvl and F3tv2 was performed by terminal primer-extension method to construct the universal plasmid standard. The expressions of F3tvl and F3tv2 in human peripheral blood monocytes and leukemia cell line THP-1 and Jurkat were validated by conceived qPCR, and the sensibility and expanded linear range were analyzed. Results F3tvl and F3tv2 were amplied specifically using the developed qPCR assay with linearity range of 101 to l0s copies per microlitre. The relative expression of F＇3tvl and was [ （5.70 × 10^-4 ） . （2.62 × 10^-5 ） ] in THP-1 cells and [ （ 1.79 × 10^-4 ） ± （4.37× 10^ - 5 ） ] in peripheral blood monocytes, while F3 tv2 were [ （ 4.94 × 10^ - 5 ） ±（ 2.19 × 10^ - 6 ） ] and [ （ 2.98± 2.18 ） × 10^ - 6 ] respectively. As negative control, no expression of both F3tvl and F3tv2 was detectable in Jurkat cells. Conclusion Human tissue factor spliced variants were specifically and sensitively quantified by using the established qPCR with universal plasmid standard. The method should be useful for providing experimental evidences in the study on ahernative splicing regulation of tissue factor.