Total RNA was first treated with DNase I to remove residual genomic DNA that might yield false-positive results during RT-PCR amplification of mRNA. Four micrograms of total RNA was incubated with 4 units of amplification-grade ribonuclease (RNase)-free DNase I (Gibco BRL) in buffer (final concentration: 20 mM Tris pH 8.3, 50 mM KCl, 2.5 mM MgCl2) in a final volume of 40 ^l for 15 min at room temperature. DNase I was inactivated by the addition of 4 ^l of 25 mM EDTA and heated to 65°C for 10 min. RNA was precipitated by the addition of 1/10 volume of 3 M NaOAc, pH 5.2, and 2 volumes of cold 100% ethanol. After 15 min at – 20°C, the samples were centrifuged at 17 000 Xg for 15 min at 4°C, and the pellet was washed with 70% ethanol before partial air drying and resuspension in 20 ^l of DEPC-treated water. Ten microliters of this final suspension was then used for cDNA synthesis. ventolin inhalers
Ten microliters DNase-treated RNA was incubated in a final volume of 20 ^l containing 10 units of placental RN-ase inhibitor, 10 mM dithiothreitol, 0.5 mM each dNTP, 50 mM Tris pH 8.3, 15 mM KCl, 3 mM MgCl2, and either 20 pmol of random hexamer primers or 500 ng of oligo-T(dT)12-18 primer.