Designing of relative quantitative Real-Time PCR for detection of CD40 gene expression in dendritic cell after suppression with antisense

Authors
Mohammad Hossein Karimi 1
Zahra Soheila Soheili 2
Ali Akbar Pourfathollah 1
Shahram Samiei 3
Seyed Mohammad Moazzeni 1
Zahra Ataee 3
Bita Geramizadeh 4
Mahnaz Kavari 3
Maryam Abdollahi 3
Padideh Ebadi 5
1 Department of Immunology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2 Department of Biochemistry, National Center Genetic Engineering and Biotechnology, Tehran, Iran
3 Iranian Blood Transfusion Research Center, Tehran, Iran
4 Transplant Research Center, Nemazi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
5 Department of Biology, Islamic Azad University, Kazeroun Unit, Kazeroun, Iran
Abstract
Objective: Dendritic cells have a critical role in control and regulation of immune responses. It is believed that these cells can be used for the treatment of many diseases. One of the methods used in immunotherapy is based on generating of tolerogenic dendritic cells through inhibition of expression costimulatory molecules. CD40 is one of the costimulatory molecules, and inhibition of expression by antisense or siRNA techniques, can generate tolerogenic dendritic cells. Generation of tolerogenic dendritic cells will be useful in the treatment of many diseases. By developing a quantitive RT-PCR for evaluation of gene expression, generation of these cells could be possible.
Using proper software we designed an Antisense and transfection of dendritic cells by lipofectamine 2000 (Invitrogen) could lead us to generate tolerogenic dendritic cells.
Materials and Methods: In this study dendritic cells were extracted from of Balb/c mice Spleen and the purity of this extraction was determined by flow cytometry. BCL1 cell line as a CD40 expressing control group and Wehi-164 cell line were cultured in RPMI-1640+10%FCS. Primer design for CD40 gene and house keeping gene (GADPH) was done by bioinformatic soft wares such as Beacon designer, mfold and Blast. RNasy plus mini kit (Qiagen) was used for RNA extraction and the Purity and integrity were determined by O.D at 260/280 and agarose gel electrophoresis. In the next step cDNA synthesized and quantitative RT-PCR for CD40 using IQ sybergreen (Biorad) were setup. Finally, standard curve for CD40 and internal control in different RNA concentrations were performed.
After transfection with lipofectamin 2000 the amount of gene suppression were quantified by qualitative RT-PCR.
Results: Using gradient real time PCR, optimum annealing temperature, Ct and ∆Rn for CD40 and GADPH were determined, annealing temperature was 59.5ºc and melting temperature was 84°c. Slope of the curve and the efficacy of PCR for CD40 and GADPH genes were quantified by serial dilution method

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