Quantitative RT-PCR Analysis of Colon Macrophages

Pieces of frozen tissue were homogenized and total RNA extracted with the help of the RNeasy plus Universal Kit (Qiagen, Hilden, Germany). To prevent inhibition of the reverse transcriptase by residual DSS present in the RNA preparation [25 (link), 26 (link)], mRNA was purified using the Dynabeads mRNA DIRECT Kit (ThermoFisher). Thereafter, the mRNA was reverse transcribed with a iScript Reaction Mix kit (Bio-Rad, Munich, Germany) followed by quantitative RT-PCR using the Power SYBR Mix (ThermoFisher). Results were normalized to the expression of the house-keeping gene HPRT and evaluated using the ΔΔCt method. In order to correct the mRNA levels of each gene for differences in macrophage numbers in the colon, the obtained values were finally normalized to F4/80 gene expression.