Comparative Analysis of Immune Gene Expression in Macrophages

GAPDH, TLR3, TLR4, IFN-β and TNF-α mRNA levels in the macrophages from naked mole rats and ICR mice were quantified by real-time PCR. Total RNA was extracted with RNAiso Plus (Takara, Dalian, China) [29 (link)] according to the manufacturer’s instructions. RNA quantity and purity were assessed by A260/A280 absorbance. First-strand cDNA was generated from total RNA with the TIANScript cDNA First-Strand Kit (Tiangen, Beijing, China). The transcript expression levels were quantified with the StepOnePlus Real-time PCR Detection System (Applied Biosystems, Warrington, UK) using the SYBR® Green RT-PCR kit (Takara, Dalian, China). Other mRNA transcript levels were then normalized to the expression of GAPDH transcripts. To allow the comparison of mRNA expression, the real-time PCR data were analyzed with the ΔΔCt method and normalized to the amount of GAPDH cDNA, the endogenous control.