Two microliters of cDNA in ribonuclease-free water was made up to 20 μl with FastStart Universal SYBR Green Master (5-carboxy rhodamine-x; 10 μl, Roche), Ultra Pure Water (6.4 μl, Sigma-Aldrich), and forward and reverse primers for AMPK-α1 and AMPK-α2. The sample was then centrifuged and 20 μl was added to a MicroAmp Fast Optical 96-Well Reaction Plate (Greiner Bio-One), the reaction plate was sealed with an optical adhesive cover (Applied Biosystems), and the plate was centrifuged. The reaction was then run on a sequence-detection system (Applied Biosystems) using AmpliTaq Fast DNA Polymerase, with a 2-min initial step at 50°C followed by a 10-min step at 95°C and then 40× 15-s steps at 95°C. This was followed by a dissociation stage with 15 s at 95°C, 20 s at 60°C, and 15 s at 95°C. Negative controls included control cell aspirants for which no reverse transcriptase was added and aspiration of extracellular medium and PCR controls. None of the controls produced any detectable amplicon, ruling out genomic or other contamination.

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