Quantitative Gene Expression Analysis in Tissues

Total RNA was extracted using the RNeasy mini kit according to the manufacturer’s instructions, which includes a DNase I treatment to remove genomic DNA (Qiagen). RNA was quantified using a Nanodrop-1000 spectrophotometer (Thermo Fisher Scientific) and stored at − 80 °C. Using the MMLV Superscript reverse transcriptase (Invitrogen) and random hexamers (Operon), reverse transcription (RT) was performed as described previously^{48 (link)}. Taqman quantitative real-time PCR (qPCR) was performed on the using the CFX96 C1000 real-time thermal cycler (Bio-Rad). The levels of gene expression were determined by normalizing to housekeeping genes, and the relative levels of the transcripts were determined by using the 2^−ΔΔCt method^{48 (link),49 (link)}. The expression levels of a ‘gene of interest’ was normalised to the geometric mean of endogenous housekeeping genes. The reference genes were selected from a panel based on their stability in R6/2 and WT samples using the Genorm kit (Primer Design) and were Hprt, Canx, and Eif4a2 for liver and Hprt, B2m and Atp5b for spleen. We did not have sufficient RNA to determine the stability of a panel of reference genes for the peripheral macrophages and therefore, these reference genes were based on previous data^{16 (link)}. Primer sequences and probes used are summarized in Supplementary Table S5 online.