Total RNA was extracted from HXO-RB44 and HEK293 using reagent (TRIzol; Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Briefly, 3 μg RNA was reverse-transcribed in a 20-μL reaction volume containing 0.5 mM dNTPs, 2.5 μM random primers, 40 U RNase inhibitor, and 200 U M-MLV reverse transcriptase (Invitrogen). Reactions were performed for 10 minutes at 25°C, 40 minutes at 37°C, and 15 minutes at 70°C, followed by cooling to 4°C. The cDNA samples were subjected to PCR using specific primers for the coxsackievirus-adenovirus receptor (CAR): 5′-TTGCTTGCTCTAGCGCTCATTGGT C-3′ (forward); 5′-TCATCACAGGAATCGCACCCATTCG-3′ (reverse). Primers for GAPDH were 5′-AATCCCATCACCATCTTCC-3′ (forward) and 5′-AGTCCTTCCACGATACCAA-3′ (reverse). PCR reaction mixtures contained 1 μL cDNA, 1.5 mM MgCl2, 0.2 mM dNTP, and 0.5 μM primers. The following reaction cycle was performed 35 times: 95°C, 30 seconds; 58°C, 30 seconds; 72°C, 45 seconds. PCR products were separated by electrophoresis on 2% agarose gels, stained with ethidium bromide, and visualized under UV light.