Gene expression in brain
Messenger RNA (mRNA) expression of inflammatory markers in the brain was determined using TaqMan® real-time PCR. The right brain hemisphere was harvested and snap frozen in liquid nitrogen. Brain hemispheres were sonicated in TRIzolTM (Life Technologies, USA), mixed with chloroform, and centrifuged at 824 x g for 15 min at 4 °C. The aqueous phase was collected and RNA was extracted using the RNeasy® Mini Kit (Qiagen, USA). RNA was quantified using a NanoDrop One spectrophotometer (Thermo Scientific, USA) and converted to 1st strand cDNA using a High Capacity cDNA RT Kit (Applied Biosystems, USA). Commercially available primers (Applied Biosystems, USA) were used to measure mRNA expression of inflammatory markers, and a house-keeping gene, GAPDH, on a CFX96 Touch Real-Time PCR Detection machine (Bio-Rad, USA). Changes in gene expression were assessed using the delta-delta CT method (Schmittgen & Livak, 2008).