For the first-strand complementary DNA synthesis, 2 lL of the viral RNA/DNA
preparation were included in a total reaction volume of 20 lL containing 4 lL of
5 reverse transcriptase (RT) buffer (50 mM Tris–HCl, 8 mM MgCl2, 30 mM KCl,
1 mM dithiothreitol pH 8.3), 0.5 mM each deoxynucleotide triphosphate (dNTP),
1 lM antisense primers of CSFV and PRRSV, respectively, 20 U of RNase inhibitor
and 5 U of avian myeloblastosis virus reverse transcriptase (Takara). After incubation
for 60 min at 42 C, the mixture was heated for 3 min at 95 C to denature
the products. The mixture was then chilled on ice.