Quantitative RT-PCR Analysis of Splenocyte Gene Expression

Splenocytes were isolated as described previously^{51 (link)}. Total RNA from splenocytes was extracted with the mini-RNA kit according to the manufacturer’s instruction (Qiagen). Using the MMLV Superscript reverse transcriptase (Invitrogen) and random hexamers (Operon), reverse transcription (RT) was performed as described previously^{52 (link)}. Taqman quantitative real-time PCR (qPCR) was performed on the Chromo4 real-time PCR detector (Biorad) as described previously and quantified via the 2^−ΔΔCT method^{52 (link),53 (link)}. The expression levels of a ‘gene of interest’ was normalised to the geometric mean of endogenous housekeeping genes (B2m and Atp5b). Primer and probe sets for Il6 and Tnfα, as well as B2m and Atp5b housekeeping genes were from Life Technologies, while real time PCR mastermix (SsoAdavanced Universal Probes Supermix) were purchased from BioRad.