4.4. Real Time RT-PCR Quantization of MCP-1 mRNA

From the LPS-stimulated THP-1cells, total RNA was isolated using TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA), and 1 µg of total RNA was used to generate complementary DNA (cDNA) using AffinityScript Multiple Temperature cDNA Synthesis Kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Real time semi-quantitative polymerase chain reaction (PCR) was carried out using TaqMan^® Fast Universal PCR Master Mix (2×) (Applied Biosystems, Foster City, CA, USA) and ViiA7 Real-Time PCR Detection System (Applied Biosystems). Human TaqMan probes and primers were purchased from Applied Biosystems: MCP-1/CCL2 (assay ID: Hs00234140_m) [18].