Gene transcripts that are important in mediating inflammatory and diabetic processes were quantified via quantitative qRT-PCR. The pro-inflammatory cytokines that were examined were TNF-α, IL-1β, and IL-6. Further, genes such as 1 L-21, IL-7, CD68, and CD14, which are important for adaptive immunity, were also quantified. In addition, two essential hormones (adiponectin and leptin) secreted by adipose tissue were also measured. β-Actin was quantified as a housekeeping gene. FASTA mRNA sequences of these mRNA transcripts were obtained from Rattus norvegicus using the National Center for the Biotechnology Information (NCBI) database. Forward and reverse primers for the genes were then generated using NCBI Primer-Blast. Primers were bought from Integrated DNA Technologies Inc (IDT), Coralville IA USA.
An iScript cDNA synthesis kit was employed to synthesize cDNAs from total RNA extracted using Rneasy Plus Universal Mini by Qiagen. The cDNAs were combined with primers and SsoFast EvaGreen supermix for the qPCR reaction using β-actin as a control transcript. The product was quantified via a Bio-Rad CFX96TM instrument (Bio-Rad Laboratories Inc.) using the manufacturer’s guidelines. The normalized relative gene expression values were determined via the delta Ct parameter.
An iScript cDNA synthesis kit was employed to synthesize cDNAs from total RNA extracted using Rneasy Plus Universal Mini by Qiagen. The cDNAs were combined with primers and SsoFast EvaGreen supermix for the qPCR reaction using β-actin as a control transcript. The product was quantified via a Bio-Rad CFX96TM instrument (Bio-Rad Laboratories Inc.) using the manufacturer’s guidelines. The normalized relative gene expression values were determined via the delta Ct parameter.