Development and validation of an efficient real-time quantitative reverse transcription polymerase chain reaction assay of Chinese goose CD4 and CD8
Zhao, QiurongChen, ShunLiu, FeiQi, YulinWang, MingshuJia, RenyongZhu, DekangLiu, MafengCheng, Anchun
Background: CD4+ T cells, which are often referred as T-helper cells, play a central role through secreting various cytokinesto enhance immune defense to pathogen. CD8+ T cells, which are called cytotoxic T lymphocytes (CTLs), provide potentdefenses against virus infection and intracellular pathogens by killing the targets cells directly. In our previous researches,the conventional and semi-quantitative PCR were used to detect the goose CD4 and CD8α. However, the semi-quantitativeRT-PCR only detect the relative amount of gene transcription. Quantitative PCR assay was more sensitive than conventionalPCR assay, and quantitative PCR assay has a lower limit of sensitivity.Materials, Methods & Results: Contrast to conventional assays, the detection of amplicons by quantitative RT-PCR couldbe visualized as the amplifi cation progressed. This effect has provided a great deal of insight into the kinetics of the reaction and it is the foundation of kinetic of real-time qPCR. The analysis of gene transcription by qPCR has proven to bean attractive method due to its potential for increasing laboratory throughput, simultaneous processing of several samplesas well as more reliable instrumentation. With those in mind, the real-time quantitative reverse transcription PCR (qRTPCR) methods for the detection of goose CD4 and CD8α transcripts were reported here for the fi rst time. With this assay,it is possible to carry out a rapid quantitative analysis of goose CD4 and CD8α transcripts over a wide linear range, withan unknown template.CD8 is expressed on the membrane of T cells either as an αα-homodimer or αβ-heterodimer. Sinceboth forms of CD8 have α chain, the transcription levels of CD8 can be monitored by detecting CD8α mRNA expression.Assays were based on the DNA sequence of goose CD4 [GenBank: JX902315], CD8α [GenBank: KC476104], and β-actin[GenBank: M26111]. qPCR was carried out in quadruplicates in a total volume of 20 µL containing...(AU)
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