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Real-Time PCR Total RNA was extracted from cells by acid guanidinium thiocyanate-phenol-chloroform extraction method, and complementary (c)DNA was synthesized using 1 μg total RNA in a 20 μL RT reaction mix containing 0.5 μg/μL of random primers, 0.1 mM dNTP, 0.1 M DTT and 5× first strand buffer