2.9. Relative transcript levels
Total RNA was extracted from cells using the TRI Reagent
(Invitrogen) and treated with RNase-free DNaseI (Fermentas) to
digest genomic DNA before use. The 5 lg treated RNA was converted
to cDNA by reverse transcription using SuperScriptTM III
First-Strand Synthesis Kit (Invitrogen). RT-PCR amplifications using
cDNAs of the respective genes were performed using corresponding
primers listed in Table 2. The PCR conditions consisted of
95 C for 3 min, followed by 30 cycles of 95 C for 15 s, 50 C for
20 s and 72 C for 20 s, and then a final extension at 72 C for
3 min. The PCR products were analyzed by 0.8% (w/v) agarose gel
electrophoresis and quantification was done using Syngene Gel
Documentation (Syngene, Frederick, MD).