Gene Expression Analysis in Brain Tissue

Total RNA was extracted from brain tissue at 3 and 7 dpi with TRIzol reagent and reverse transcribed to cDNA using a RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, Vilnius, Lithuania) (Yang et al., 2008 (link)). The PCR reaction (35 cycles) was performed using a DreamTaq Green PCR Master Mix Kit (Thermo Scientific). Quantitative RT-PCR was performed using the following forward and reverse primer sets designed using Premier Biosoft 5 (Palo Alto, CA, USA): TNF-α, 5′-ATA AGA GCA AGG CAG TGG AG-3′ and 5′-TCC AGC AGA CTC AAT ACA CA-3′;IL-6, 5′-AGC CAG AGT CCT TCA GAG AG-3′ and 5′-TCC TTA GCC ACT CCT TCT GT-3′; IL-10, 5′-TTC TCA TTC CTG CTT GTG GC-3′ and 5′-ATC TGA GTG TGA GGG TCT GG-3′; Bax, 5′-CTG ACA TGT TTT CTG ACG GC-3′ and 5′-TCA GCC CAT CTT CTT CCA GA-3′; Bcl-2, 5′-CGC TGG GAG AAC AGG GTA-3′ and 5′-GGG CTG GGA GGA GAA GAT-3′; Caspase-3, 5′-AGA TAC CGG TGG AGG CTG ACT-3′ and 5′-TCT TTC GTG AGC ATG GAC ACA-3′; and β-actin (control), 5′-GGG AAA TCG TGC GTG ACA T-3′ and 5′-TCA GGA GGA GCA ATG ATC TTG-3′. Products were resolved by 1.5% agarose gel electrophoresis with ethidium bromide staining. The mRNA levels of TNF-α, IL-6 and IL-10 were detected at 3 dpi, and those of Bax, Bcl-2, and caspase-3 were detected at 7 dpi. Quantitative analysis was performed using a Tanon 4100 Gel Imaging System (Tanon Science & Technology Co., Shanghai, China).