2.10 RNA preparation and RT-PCR
Total RNA of brain tissues was isolated by using Trizol (Takara,
Kusatsu, Japan) and then reverse-transcribed to cDNA using
PrimeScriptTM RT Master Mix Kit (Takara). RT-PCR was
conducted with SYBR Green master mixes (Takara) on Roche LightCycler480
System to measure mRNA levels of Trpm4 , interleukin
(IL)-1β , tumor necrosisfactor-α (TNF-α) , inducible
nitricoxide synthase (iNOS) , transforming growth factor-β
(TGF-β) , Arg1 , IL-10 and glyceraldehyde phosphate
dehydrogenase (Gapdh) . Relative changes of mRNA expressions were
normalized to Gapdh [19].