A Pure Link RNA Mini kit (Invitrogen, Carlsbad, CA, USA) was used for the extraction of total RNA from HUVECs, and cDNA was synthesized with a Superscript VILO cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) on a CFX connect thermal cycler (Bio-Rad, Hercules, CA, USA). The value of each cDNA was calculated using the ΔΔCq method and normalized to the value of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Oligonucleotide PCR primers targeting MCP-1 mRNA were designed according to a previous report [44 (link)], and the specificity of the primers was confirmed by BLAST search and melting curve analysis. The primer sequences are shown inTable 1 .
The reaction conditions were as follows: activation step at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min.
Oligonucleotide PCR primers targeting MCP-1 mRNA were designed according to a previous report [44 (link)], and the specificity of the primers was confirmed by BLAST search and melting curve analysis. The primer sequences are shown in
The reaction conditions were as follows: activation step at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min.
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