Development and Validation of an Effi cient Real-time Quantitative Reverse Transcription Polymerase Chain Reaction Assay of Chinese Goose CD4 and CD8α
Year: 2014
Type: article
Abstract: "Background : CD4+ T cells, which are often referred as T-helper cells, play a central role through secreting various cytokines to enhance immune defense to pathogen. CD8+ T cells, which are called cytotoxic T lymphocytes (CTLs), provide potent defenses 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 CD8 α . However, the semi-quantitative RT-PCR only detect the relative amount of gene transcription. Quantitative PCR assay was more sensitive than conventional PCR 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 could be visualized as the amplifi cation progressed. This effect has provided a great deal of insight into the kinetics of the reac- tion and it is the foundation of kinetic of real-time qPCR. The analysis of gene transcription by qPCR has proven to be an attractive method due to its potential for increasing laboratory throughput, simultaneous processing of several samples as well as more reliable instrumentation. With those in mind, the real-time quantitative reverse transcription PCR (qRT- PCR) methods for the detection of goose CD4 and CD8 α 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 CD8 α transcripts over a wide linear range, with an unknown template.CD8 is expressed on the membrane of T cells either as an αα -homodimer or αβ -heterodimer. Since both forms of CD8 have α chain, the transcription levels of CD8 can be monitored by detecting CD8 α mRNA expression. Assays were based on the DNA sequence of goose CD4 [GenBank: JX902315], CD8 α [GenBank: KC476104], and β -actin [GenBank: M26111]. qPCR was carried out in quadruplicates in a total volume of 20 μL containing 10 μL Ssoadvanced™ SYBR®, 1.5 μL of cDNA, 0.6 μl of each primers (10 μM), and 7.3 μL of sterile water. The amplifi cation program was 94°C for 3 min, followed by 40 cycles of 94°C for 10 s, the annealing temperature of each pair primers for 30 s. The annealing temperatures for real-time qPCR were 61.4°C for CD4 and CD8 α , 60°C for β -actin, respectively. After amplifi cation, melting analysis of qPCR revealed specifi c amplifi cation of each gene that could be visualized clearly as a single peak of melting temperature. The R2 values of the standard curves for all target genes were above 0.999. To further apply the methods we established, the specifi c real-time qRT-PCR methods were performed for the detection of the biology activity of CD4 and CD8 α in the spleen mononuclear cells (MNCs). Fresh spleen monocytes cells (MNCs) were chosen as the responder, PHA was chosen as agonist, and the transcripts of CD4 and CD8 α mRNA in spleen MNCs after stimulation were investigated by qRT-PCR assay. Results"
Institution: Sichuan Agricultural University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: diamond
OpenAlex ID: W1521728862