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In general, PCR reactions were performed in a volume of 50 μl with 50 picomoles of each primer, dNTP's (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) at a final concentration of 0.5 mM, 1 μl of template, 1X PCR buffer (10X PCR buffer contained 700 mM Tris pH 8.8, 20 mM MgCl 2 , 1% Triton X-100, and 0.1% Tween 20) and Taq polymerase (Perkin-Elmer Inc., Wellesley MA) diluted to the manufacturers recommendations.
PCR amplification was performed, with final concentration of the digest equivalent to approximately 100 ng of the DNA, 1 × PCR bufferII (Perkin-Elmer, Foster City, CA, USA), 2.5 mmol/l MgCl 2 , and amplitaq gold 1.25 units.
Reaction conditions were as follows for a 50 μl PCR reaction : Perkin-Elmer Cetus PCR buffer; 1.5 mM MgCl 2 , 0.25 mM dNTPs, 1.5 μl of each primer (10 μM), 1% DMSO, 5 U of Taq polymerase (Life Technologies).
Confirmation by Taqman (Perkin-Elmer) quantitative PCR was performed for nine selected genes as described in [ 12].
The structure of all reporter constructs were confirmed by DNA sequencing (Amplitaq sequencing kit, Perkin-Elmer).