Purinergic P2Y Receptor RT-PCR

Purinergic P2Y receptor (P2RY) expression levels were determined by RT-PCR. Total RNA was isolated from THP-1 cells and primary human monocytes with the NucleoSpin RNA II Kit according to the manufacturer’s instructions, and reverse transcription was performed as described before (11). 10 ng of corresponding cDNA were subjected to amplification by PCR [10 min 95°C, 33x (15 s 95°C, 1 s 60°C)] with distinct primer pairs for each P2RY subtype (Supplementary Table 1). Human genomic DNA was used as positive and H₂O as negative control. Amplification products were analyzed by agarose gel electrophoresis (3% agarose gel).