Measurement of mRNA expression using qRT-PCR

Total RNA was isolated from splenocytes cultured as described above and then cDNA was prepared from RNA using iScript cDNA synthesis kit. The resulting cDNA was amplified in an Applied Biosystems StepOne instrument using SYBR Green PCR Master Mix and appropriate primers. The sequences of the forward and reverse primers for the detection of mRNAs were selected from the Primer Depot (National Institutes of Health (NIH)) and the corresponding primers were then obtained from Sigma-Aldrich. The mRNA levels of specific genes were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA levels, and the relative gene expression levels were determined. The results were expressed either as ‘Fold change’ or ‘Relative message’. The optimal time point of expression of individual genes was used for further characterization.