2.2. cDNA Synthesis

The isolated RNA was used to synthesize cDNA employing a SuperScript II kit (Invitrogen, Carlsbad, California) according to the manufacturer’s instructions. First, random hexamer primers were added to each 5 μL (RNA plus DNA) sample to a concentration of 6.5 ng/μL. Each sample was then mixed gently, incubated at 70 °C for 10 min, and then chilled on ice. The first strand synthesis consisted of 33.3 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl₂, 6 mM DTT (dithoithreitol), 330 μM dNTPs (82 μM of each dATP, dCTP, dGTP, dTTP), and 100 units of SuperScript II reverse transcriptase (final volume of 15.5 μL) for each sample; incubated at 37 °C for 90 min and then placed on ice. Second strand synthesis consisted of 25 mM Tris-HCl (pH 6.9), 93 mM KCl, 4.6 mM MgCl₂, 140 μM β-NAD⁺, 9.3 mM (NH₄)₂SO₄, 0.5 mM dNTP mix (as above), 1.2 mM DTT, 5 U Escheria coli DNA ligase, 20 U E. coli DNA polymerase I, and 1 U E. coli RNase H (in a total of 80 µL); incubated for 2.5 h at 15 °C. Next, 5 U T4 DNA polymerase was added to each sample and incubated at 15 °C for 7 min, and then placed on ice. The reaction was stopped by the addition of EDTA to a concentration of 30 mM. Then, each sample was subjected to chloroform/isoamyl alcohol (24:1) treatments, centrifuged at 14,000× g for 2 min to separate the layers, and the nucleic acids (aqueous phase) were precipitated in 0.5 M NaCl and 80% ethanol. Precipitation was performed at −20 °C for 15 min. After centrifugation at 14,000× g for 20 min, the pellets were washed with -20 °C 80% ethanol, and centrifuged at 14,000× g for 2 min. After decanting, the pellets were dried in a vacuum centrifuge drying (Eppendorf Vacufuge, Hauppauge, NY) at 45 °C for 10 min, and then were rehydrated in 9 μL of DEPC-treated water. These were mixed with the corresponding DNAs.