Abstract:

BACKGROUND: The inducible costimulatory molecule (ICOS) / its ligand (ICOSL) become more and more attractive in the field of anti-infection, anti-transplantation reaction, anti-tumor and treatment of autoimmune disease. Current studies focus on molecular level of ICOS/ICOSL gene expression, no study reports quantitative detection of ICOS/ICOSL mRNA expression.
OBJECTIVE: To construct the plasmid standard samples of ICOS/ICOSL cDNA, and to detect the ICOS/ICOSL gene expression by the Taqman Probe technology.
METHODS: The cDNA was synthesized by reverse transcription with Random 9 mers as the primer and total RNA from the gastric adenocarcinoma tissues as the template. The target sequences of human ICOS/ICOSL cDNA were amplified by polymerase chain reaction amplification from the resulting cDNA as mentioned above. The PMD-18-ICOS/ICOSL recombinant plasmids were constructed. Standard curve was made by Taqman Probe fluorescence polymerase chain reaction technology after gene sequencing.
RESULT AND CONCLUSION: The composition of total RNA extracted from the tissues was complete and the sequences of the ICOS/ICOSL plasmid were consistent with the target fragments. The initial concentration of plasmids was 6.10×10¹³, 4.31×10¹³ copies/mL. The different diluted concentration of the plasmids had good linear relationship (R²=1) after amplifying by Taqman PCR and the linear range was 10¹²-10¹³ copies/mL, suggesting that the method which can quantitatively detect the concentration of ICOS/ICOSL gene by Taqman Probe technology was successfully constructed.