Quantifying Gene Expression in Immune Cells

2.5 µg total RNA of each spleen was subjected to a reverse transcription reaction in a total volume of 40 µl employing 5 × FS-Buffer, 3 µg random hexamer primer, 200 U M-MLV transcriptase, 0.02 µmol dNTPs, and 0.4 µmol DTT (all Invitrogen^TM GmbH) as well as 40 U RNAse inhibitor (Agilent Technologies). 70 °C denaturation step was followed by a 1.5 h 42 °C reverse transcription step. 1 µl of individual cDNA preparations from each IL-10R-blocked and isotype-treated mouse, respectively, was pooled. The two pools were diluted 1:200 for subsequent qPCR which was performed as described previously^{47 (link)}. Transcripts of three housekeeping genes and 32 genes involved in cytokine-, interferon-, chemokine- and innate immunity-related signaling were quantified as fold changes using the ΔΔC_T-method. For a detailed list of genes and primer sequences, see supplemental Table S2. Transcripts reaching a threshold fold change of 1.5 were regarded as potentially regulated and subjected to RT-qPCR in non-pooled samples.