Designing Specific Primers for Amplification and Quantitative Analysis of the GPR۱۲۰ and PPARγ Genes

Background: Designing the primer pairs is one of the most important factors affecting the amplification and quantitative analysis of the nucleic acid sequences of interest. Using in silico methods, the present study intends to design highly specific primers for quantitative analysis of the genes having very low expression. To achieve this aim, we selected two candidate genes having very low expression, namely, G-protein coupled receptor ۱۲۰ (GPR۱۲۰) and peroxisome proliferator-activated receptor-g (PPARg), in peripheral blood leukocytes of healthy volunteers. Materials And Methods Peripheral blood was collected from ۳۰ healthy volunteers. Primers for GPR۱۲۰ and PPARγ were designed using online websites (UCSC, Oligocalc, and Oligoanalyzer) and the primer designing tool (NCBI). Total RNA extraction and cDNA synthesis were done using commercially available kits based on the instructions of the manufacturers. Finally, the melting curve analysis of GPR۱۲۰ and PPARγ was assessed using the quantitative real-time PCR method. Results: The investigation of the in silico gene expression revealed that GPR۱۲۰ and PPARγ have very low expression in leukocytes. Besides, the melting curves analysis for GPR۱۲۰ and PPARg genes in the studied individuals showed only one melting peak, confirming the specific amplification of the desired genes. Conclusions: Altogether, the study findings indicated that we can utilize the peripheral blood sample for assessing the gene expression and amplification of omega-۳ fatty acids receptors, i.e. GPR۱۲۰ and PPARg as two candidate genes possessing very low expression in leukocytes.

Keywords:

GPR۱۲۰ , Omega-۳ fatty acid , cDNA , PPARγ , Real-time PCR